Chemotherapy-induced adipo-lineage cell senescence drives bone loss

Domain: biology
Published: 2025-12-30
Editorial outcome: major_revision

Abstract
Chemotherapy-induced bone loss is a debilitating and common side effect of cancer treatment, though its underlying mechanisms remain poorly understood. Here, we show that, despite the systemic administration of chemotherapy, cellular senescence is restricted to bone marrow adipo-lineage cells specifically Cxcl12-abundant reticular (CAR) cells and bone marrow adipocytes (BMAds). Induction of senescence within these populations promotes RANK ligand (RANKL)-mediated osteoclastogenesis, leading to significant bone loss. Notably, we find that inhibition of the p38 MAPK-MK2 pathway suppresses the senescence-associated secretory phenotype (SASP), including RANKL production abrogating bone loss. Furthermore, treatment with the senolytic combination dasatinib and quercetin (D+Q) selectively eliminates senescent CAR cells and BMAds, effectively preventing chemotherapy-induced bone loss. Given that nearly all chemotherapy treated patients experience bone loss and associated fracture risk, our findings offer a promising therapeutic avenue to preserve bone integrity and improve quality of life for cancer patients.

Introduction
Chemotherapy remains a major weapon in our anti-cancer armamentarium. However, while chemotherapy can slow tumor progression and in some instances be curative, it also drives a wide range of comorbidities that can significantly impact a patient’s quality of life and in some instances, be so severe that it results in dose-de-escalation that negatively impacts its anti-tumor properties and patient survival1,2. One such side effect is bone loss, which occurs in nearly all patients treated with chemotherapy. Patients who experience substantial bone loss are more susceptible to pain and fracture and patients that experience a fracture have reduced overall survival rates3.

Senescent cells undergo cell cycle arrest and typically express p16, senescence-associated-β-galactosidase (SA-β-gal), and senescence associated secretory phenotype (SASP) factors that are post-transcriptionally regulated by the p38 mitogen-activated protein kinase-MAPK-activated protein kinase 2 (p38MAPK-MK2) pathway4. The SASP consists of a plethora of cytokines, chemokines, growth factors and factors that remodel the extracellular matrix (ECM) and impact numerous cells including immune cells5. We previously reported that the elimination of senescent cells or inhibition of SASP production using genetic and pharmacological approaches to inhibit the p38MAPK-MK2 pathway prevented chemotherapy-induced bone loss6. However, the cells and mechanisms responsible remained elusive. Given a growing number of reports that the cell type undergoing senescence expresses unique SASP factors that can have a profound and specific impact on tissue homeostasis7, our previous report left a number of critically unanswered questions. Further, the type of experienced stress can also dictate what cell types enter senescence. Thus, it was interesting to note that in the context of aging, senescent osteocytes were found to contribute to bone loss8, and our studies failed to implicate osteocytes in chemotherapy-induced bone loss6.

Results
Chemotherapy induces senescence and impacts bone homeostasis
Previously we demonstrated that several weekly doses of doxorubicin led to senescence and bone loss6. While the long-term damaging effects of chemotherapy on bone health are well known9, it is unclear whether a single dose of chemotherapy is sufficient to induce senescence and drive bone loss. To address this question, we established a 9-day model (Fig. S1A). 12-week-old female mice (C57BL/6) were treated with either doxorubicin (Doxo) or Paclitaxel (PTX) and we used 3D μCT to examine bone density as measured by bone volume over total volume (BV/TV) on day 9. We found that a single 5 mg/kg dose of Doxo was sufficient to cause significant bone loss (Fig. S1B, C). In addition to bone loss, other bone parameters were also disrupted including a drastic reduction in trabecular number and thickness and increases in trabecular spacing was noted in chemotherapy-treated mice compared to vehicle (Veh) treated mice with unchanged cortical bone thickness (Fig. S1D–S1I). Similar results were observed when mice received clinically relevant PTX (Fig. S1J–S1Q). These findings were not limited to female mice, Doxo also induced bone loss in male mice (Fig. S1R–S1Y). Because bone volume is maintained by balanced osteoclast and osteoblast activity, we also interrogated them. Tartrate resistant acid phosphatase (TRAP), a marker of osteoclasts, staining on the femur of Doxo-treated mice showed significantly more differentiated osteoclasts that covered more bone surface (Fig. S2A–S2C). Using the Col1a1-GFP mouse that allows one to directly measure osteoblasts on the bone surface, we also found that osteoblast numbers were reduced in Doxo-treated mice (Fig. S2D, S2E) compared to Veh-treated mice. To assess osteoblast activity, we treated mice with calcein and alizarin to assess bone formation. Using this strategy, we found that the bone formation rate (BFR/BS) was significantly decreased in Doxo-treated mice relative to Veh-treated mice after 9 days (Fig. S2F, S2G). Because the bone mineralization rate is dependent on collagen deposition, we also assessed collagen type-1 (Col1a1) production by quantitative RT-qPCR in crushed bone from which bone marrow was largely removed by centrifugation (hereafter termed bone-resident fraction), which is enriched in osteoblasts. We found that Col1a1 was significantly reduced upon Doxo treatment (Fig. S2H), raising the possibility that its reduction contributed to reduced mineralization rate. Collectively, these results demonstrated that a single dose of chemotherapy is sufficient to induce rapid bone loss and shifts the balance of bone remodeling toward bone resorption at the expense of bone formation.

To determine if senescence contributed to the rapid bone loss observed after a single dose of chemotherapy, we treated 12-week-old INK-ATTAC mice, that express a p16INK4a dependent inducible suicide gene10 with Doxo followed by Veh or AP20187 (AP, 10 mg/kg) on days 2, 4, 6, and 8 to eliminate p16+ senescent cells (Fig. 1A). To determine if senescence was induced in this time frame and if AP could eliminate p16+ senescent cells, we performed senescence-associated beta-galactosidase (SA-β-gal) and p16 protein staining on treated bones and found that both were increased upon Doxo treatment and eliminated in the femurs of mice treated with AP (Fig. 1B–E). To ensure specificity of our p16 antibody we also stained bones from p16 knockout animals treated with Doxo and failed to detect p16 protein (Fig. S2I). We also evaluated Cdkn2a (p16) gene expression in the Doxo-treated bone-resident fraction. We found elevated levels of Cdkn2a mRNA in the bone-resident fraction that was abolished by AP treatment (Fig. 1F). To ascertain the impact of a single dose of chemotherapy on bone parameters, we subjected the femurs to μCT. We found that AP treatment of Doxo-treated mice restored trabecular bone density (Fig. 1G-I), bone mineral density (BMD) and trabecular thickness (Tb.Th) without significantly impacting other bone parameters (Fig. S3A–E). Similar results were obtained with PTX treatment (Fig. S3F–K).

Bone loss can result from increased osteoclastogenesis and/or decreased osteoblastogenesis. To ask if senescence impacted osteoclasts, we stained the femurs of treated mice for TRAP. As expected, we found that Doxo treatment increased osteoclast numbers compared to Veh-treated mice. When we examined the femurs of INK-ATTAC mice treated with Doxo + AP, we found that osteoclast numbers and coverage of bone were similar to Veh-treated mice (Fig. 1J–L). Next, we assessed the impact of chemotherapy on osteoblast numbers. For these analyses, we utilized osteocalcin staining (OCN+ cells) to quantify osteoblasts. Femurs were isolated and sections stained with OCN. When we quantitated OCN+ cell numbers on trabecular bone, we found they were decreased in the femurs from Doxo-treated mice and this was reversed upon AP treatment (Fig. S3L, M). We also assessed bone formation rates by treating INK-ATTAC mice with calcein and alizarin to assess bone formation rates and we found that the reduction in bone formation noted in Doxo-treated mice was restored by AP treatment (Fig. S3N, O). Taken together, these findings indicate that chemotherapy-induced senescence is associated with rapid disrupted bone homeostasis that leads to bone loss.

Senescent bone resident cells drive chemotherapy-induced bone loss
Chemotherapy is a systemic treatment that impacts cells across the body, and bone homeostasis can be impacted by local bone resident cells and systemic signaling pathways11. To ask if bone resident cells drove bone loss, we employed a vossicle model system that allows one to transplant vertebral bodies from one syngeneic mouse into a recipient mouse allowing us to mix genotypes12,13. For these studies, the L4 and L5 vertebrae from 4-day-old neonate INK-ATTAC mice were implanted in wild-type 10-week-old C57BL/6 syngeneic recipient mice to ask if senescent bone resident cells were responsible for chemotherapy-induced bone loss. Two weeks after the implantation of the vertebral bodies, when a blood supply was established14, mice were injected with 5 mg/kg doxorubicin (Doxo) weekly for 4 weeks (Fig. 2A, B). To assess bone loss, the vertebral bodies and femurs were removed from the mice 9 days after the final Doxo dose and bone density was assessed by μCT. Analyses of the vertebrate by μCT revealed that AP treatment prevented Doxo-induced bone loss in implanted INK-ATTAC vertebral bodies (L4 & L5) (Fig. 2C and S4A). In contrast, bone loss in recipient wildtype mouse femurs was not rescued by AP treatment (Fig. 2D). Furthermore, quantification of TRAP+ osteoclasts in the vertebral bodies revealed that AP treatment also abrogated the increased osteoclast numbers in INK-ATTAC vertebrate (Fig. 2E–G, S4B–D) but failed to reduced TRAP+ osteoclasts in recipient wildtype mouse femurs (Fig. S4E–G). Taken together, these findings indicate that senescent bone resident cells drive chemotherapy-induced bone loss.

Vertebrate contain CD45+ stem cells and immune cells including T cells and macrophages, which can contribute to bone homeostasis15 and can undergo senescence16,17. Thus, we next asked if senescent CD45+ cells contributed to chemotherapy-induced bone loss by carrying out a bone marrow transplant using bone marrow from INK-ATTAC mice into wildtype mice. This allowed us to induce the Cdkn2a-driven suicide gene in senescent CD45+ derived cells exclusively. Recipient wildtype C57BL/6 (CD45.1) mice were treated with two doses of 400 cGy 4 hours apart and then received bone marrow (5 × 106 cells/200 μl/mouse) from INK-ATTAC mice (CD45.2) and animals were allowed to recover for 6 weeks (Fig. S5A). To assess chimerism, peripheral blood mononuclear cells (PBMC) from recipient mice were stained with an anti-CD45.2 and anti-CD45.1 antibody and we found that mice obtained greater than 90% chimerism (Fig. S5B, C). Recipient mice were then treated with PTX ± AP and 9 days later bone density was assessed by μCT. We found that AP-treatment failed to prevent PTX-induced bone loss (Fig. S5D), indicating that p16 positive senescent bone marrow-derived CD45+ cells did not contribute to chemotherapy-induced bone loss.

Chemotherapy induces senescence in bone marrow adipo-lineage cells
Farr et al. recently demonstrated that mouse osteocytes (OCY) undergo senescence with aging and contribute to bone loss8. However, we failed to detect elevated levels of Cdkn2a (p16) gene expression in OCY-enriched bone fractions as evidenced by DMP expression (Fig. S5E) from mice treated with Doxo (Fig. S5F), indicating that chemotherapy does not induce senescence in osteocytes.

To identify which bone resident cells senesced in response to chemotherapy, we performed single-cell RNA-sequencing (scRNA-Seq) on cells isolated from the bone marrow following MACs depletion of CD45+, CD71+, and Ter119+ cells (referred to as the bone resident enriched fraction, Fig. 3A). Our transcriptomic profiling identified a total of six clusters in both Veh- and Doxo-treated mice (Fig. 3B, C): Cxcl12-abundant reticular (CAR) cells, immature osteoclasts, two fibroblast populations, osteoblasts, and chondrocytes, as defined by distinct gene expression patterns (Fig. S5G). Next, we performed a gene set enrichment analyses (GSEA) and found that when we compared the different Doxo-treated cell populations to their corresponding Veh-treated population, the only one that showed an enrichment was the CAR cell population. Indeed, the Doxo-CAR cell population showed an enrichment of the CHICAS_RB1_TARGETS_Senescence and the FRIDMAN senescence signature (Fig. 3D, E). Based on the CHICAS_RB1_TARGETS gene set, we found that only the Doxo-treated CAR cells exhibit a senescence-associated gene signature compared to Veh-treated CAR cells as well as other Doxo- and Veh-treated clusters (Fig. 3F, S5H). Next, to determine which CAR cells expressed p16, we sub-clustered the CAR cells and found 3 sub-populations (Fig. 3G). Among them, only the CAR cell population 1 exhibited elevated Cdkn2a expression levels and reduced levels of the cell proliferation marker (Mki67) in the Doxo group (Fig. 3H). Furthermore, we analyzed a publicly available human bone scRNA-seq dataset (GEO: GSE230295) derived from acute lymphoblastic leukemia patients receiving NOPHO2008, an aggressive chemotherapeutic regimen that includes anthracyclines (e.g. daunorubicin or doxorubicin)18. Our transcriptomic profiling identified a total of seven distinct clusters (Fig. S5I): two T cell populations, hematopoietic stem cells (HSCs), CAR cells, B cells, and two erythroid progenitor populations, each defined by characteristic gene expression profiles (Fig. S5J). Gene set enrichment analysis (GSEA) revealed that the CAR cell population was enriched for the FRIDMAN senescence signature (Fig. 3I). Consistent with this, senescence-associated signatures were most prominent in the CAR cell population, which displayed elevated expression of Cdkn2a, Cdkn1a, and Glb1, along with reduced levels of the proliferation marker Mki67 (Fig. 3J) compared to other cell populations. This analysis demonstrates that chemotherapy induces senescence in human CAR cells.

To confirm that p16 was increased in CAR cells, we turned to a senescence reporter mouse. For these studies, we utilized the recently characterized p16-CreERT2/tdTomato mouse in which the first exon of the endogenous p16INK4a gene was substituted with a CreERT2 gene, which allowed us to identify p16 expressing cells (tdTom+) at the single-cell level19. For these studies, 12-week-old p16-CreERT2/tdTomato mice were treated with either Doxo or PTX and 9 days later we assessed tdTom+ cells in the femurs. As expected, we found tdTom+ cells present in the marrow space of the femurs and failed to see tdTom+ OCY (Fig. S6A). Analysis of the femurs revealed the increased tdTom+ cells in the metaphysis region of the femur of Doxo and PTX treated mice (Fig. 4A, B, S6B, S6C). To confirm that these p16 positive cells were senescent, we assessed SA-β-gal by using the fluorescent SPiDER-β-gal probe. We found that PTX-treated mice contained increased numbers of double-positive SPiDER+; tdTom+ cells (Fig. S6D), indicating that they were senescent.

To determine if p16 was expressed in CAR cells, we first enriched for CAR cells by sorting CD45-Ter119-CD31-Sca1-Pdgfrβ+ cells using bone tissue including marrow as previously described20 (Fig. S6E) and carried out RT–qPCR analysis. We found that Doxo treatment increased Cdkn2a expression in CAR cells compared to CAR cells isolated from Veh-treated mice (Fig. 4C). We also noted that the tdTom+ signal was associated with both smaller cells and a large ring-like structure that was reminiscent of mature adipocytes (BMAds) (Fig. S6B). To ask if BMAds, which would not survived the isolation that we performed for our scRNA-seq and thus were absent in the analysis, were senescent, we used an approach that allows you to isolate them without destroying them21,22. Using this approach, we isolated BMAds and examined their gene expression by RT-qPCR. As expected BMAds expressed significant levels of Cdkn2a (p16), Cdkn1a (p21), and Il6 following Doxo treatment (Fig. 4D–F) indicative of senescent BMAds (senBMAds). Next, to ask if these cells were important in our model, we treated INK-ATTAC mice with Doxo or Veh ± AP and carried out SA-β-gal/oil red O co-staining and immunostaining. While we observed no difference in p16+/PPARγ- cells, Doxo treatment increased p16+/PPARγ+ cells that were decreased upon AP treatment in INK-ATTAC mice (Fig. 4G, H, S6F). Further, we found that the femurs from Doxo-treated mice also contained increased numbers of SA-β-gal/oil red O-double positive cells that was again abrogated by AP treatment (Fig. S6G, S6H). Next, confirm that p16 was absent in fibroblasts and chondrocytes after 9 days of Doxo treatment, we used the p16-CreERT2/tdTomato mouse and stained with antibodies for fibroblast (S100a4) and chondrocyte (Sox9) specific markers. We failed to find colocalization of p16-driven tdTom with S100a4 (Fig. S6I) or Sox9 (Fig. S6J). Additionally, we did not observe p16-driven tdTomato signal on the bone surface, indicating that osteoclasts and osteoblasts did not undergo p16-related senescence (Fig. S6K, L).

Our scRNA-Seq suggested that CAR cells were senescent and this was supported by our finding that tdTom+ cells were present in the marrow of Doxo-treated mice (Fig. 4A). To confirm that CAR cells underwent senescence, we used the Cxcl12-driven GFP reporter mouse and stained bone sections for p16 following Doxo treatment. We found that GFP and p16 colocalized in Doxo-treated mouse femurs (Fig. S6M, N). We also stained femurs from p16-CreERT2/tdTomato mice for EBF3, a transcription factor preferentially expressed by CAR cells20. We found that both Doxo and PTX treatments led to an increase in the number of EBF3+; tdTom+ as well as EBF3-; tdTom+ cells (Fig. 4I, J, S6O). A similar pattern was observed with PTX treatment (Fig. S6P, S6Q), suggesting that the EBF3-; tdTom+ population includes mature adipocytes that undergo senescence in response to these chemotherapeutic agents. Finally, to confirm these findings in the INK-ATTAC model we stained for EBF3 and SPiDER (the antibodies for EBF3 and p16 are the same species so we did not use p16 for these stains). We found that Doxo-induced EBF3+; SPiDER+ cells were reduced in INK-ATTAC mice that were also treated with AP (Fig. 4K, L) with no changes in EBF3-; SPiDER+ cells in Veh and Doxo groups (Fig. S6R). Together, these data argue that chemotherapy-induced p16+ senescence was restricted to adipo-lineage cells.

Senescent adiponectin positive cells drive chemotherapy-induced bone loss
Having demonstrated that CAR cells and BMAds senesce in response to chemotherapy, we next asked if they were sufficient to drive chemotherapy-induced bone loss. To test this, we crossed adiponectin-Cre (ADQ-Cre) mice, which targets CAR cells and BMAds23 to mice bearing the diphtheria toxin receptor (DTR), downstream of a floxed stop codon, creating ADQ-Cre/DTR mice. Diphtheria toxin (DT) injection is non-toxic in wildtype mice and exclusively targets cells expressing the DTR. To characterize the ADQ-Cre/DTR mice, 11-week-old mice were treated with DT (100 ng/mouse/day) for 5 consecutive days (Fig. S7A). DT injection led to efficient ablation of epididymal white adipose tissue (eWAT) and the bone marrow-associated adipocytes (BMAds) (Fig. S7B–D) in ADQ-Cre+ mice, but not in Cre- mice as expected. To assess the role of CAR cells and adipocytes in chemotherapy-induced bone loss, mice received DT followed by a single dose of Doxo (Fig. S7A). Nine days later, we stained bone sections to evaluate p16+ cells. As expected, there was no difference in the numbers of p16+ cells in the Veh- versus Doxo-treated ADQ-Cre+/DTR mice (Fig. S7E and S7F). Subsequently, we performed μCT analysis of the femurs. As reported early, ADQ-Cre+/DTR mice treated with Veh showed a significant increase in femur trabecular bone (BV/TV)24. Surprisingly, ADQ-Cre+/DTR mice failed to lose bone following Doxo treatment in both female (Fig. 5A, B, S7G–J) and male mice (Fig. S7K, L) with no changes in cortical bone thickness in female mice (Fig. S7M). Given that the ADQ-Cre+/DTR mouse displays increased baseline BV/TV and all adipocytes throughout the mouse are lost, we created a mouse model that allowed us to eliminate only p16+ senescent adiponectin-positive cells. To accomplish this, we cloned a lox-stop-lox cassette 5’ of the INK-ATTAC allele followed by a P2 cleavage site and tdTomato (Fig. S8A). This construct was knocked into the ROSA26 safe harbor locus and the resulting QR mouse was mated to the ADQ-Cre mouse. When the ADQ-Cre/QR mouse was treated with Doxo and AP, EBF3+; SPiDER+ cells were eliminated (Fig. S8B, C). μCT analysis demonstrated that targeting these p16+ adiponectin positive cells effectively prevented chemotherapy-induced bone loss (Fig. 5C, D, S8D–G). These data demonstrate that adiponectin positive senCAR cells and senBMAds drive chemotherapy-induced bone loss.

Previously we demonstrated that chemotherapy altered osteoclast and osteoblast numbers (Fig. 1J–L, S3L and S3M). To ask if adipocyte ablated mice restored these numbers, we assessed the osteoclast population in DT-treated ADQ-Cre+/DTR mice and found that osteoclast numbers remained unchanged between the Veh- & Doxo-treated groups (Fig. S8H–J). Similarly, osteoblast numbers were also maintained in response to Doxo (Fig. S8K, L). Taken together, these results demonstrated that BM adipo-lineage cells drive chemotherapy-induced bone loss by altering osteoclast and osteoblast numbers.

Given CAR cells, which are OB and adipocyte progenitors undergo chemotherapy induced senescence, we next questioned if this would result in reduced OB differentiation. To address this, bone marrow cells were isolated from Doxo- and Veh-treated mice nine days after treatment, plated at the same density, and cultured in osteoblastic differentiation medium for up to 21 days as previously described25. We found that despite plating the same number of bone marrow cells (1×104 cells/per well), cells from Doxo treated mice had reduced osteogenic differentiation potential at day 21 (Fig. S9A). In addition, ALP activity at day 14 (Fig. S9B) and mineralization capacity as assessed by alizarin red S staining at day 21 (Fig. S9C) were dramatically reduced. The reduction in mineralization was consistent with our in vivo findings (Fig. S3N, S3O). While perhaps not surprising given the precursors are sensing in response to chemotherapy, these results demonstrate that osteoblastic differentiation from Doxo-treated bone marrow cells is severely compromised. To ask if adipocyte differentiation was also impacted by Doxo, we cultured bone marrow cells from Doxo-treated mice in adipogenic medium. After 14 days, we found that adipogenic differentiation was reduced (Fig. S9D, E).

Senescent BM adipo-lineage cells drive bone loss through increase expression of RANKL
Given the significant increases in osteoclasts following chemotherapy treatment, we wanted to ask if senescent CAR cells and adipocytes were responsible for osteoclast differentiation. In the current study we showed that osteocytes (OCY) do not senesce in response to chemotherapy, however they can regulate osteoclastogenesis through RANKL (Tnfsf11) secretion26. To ask if senescent CAR cells/BMAds drove osteocytes to express, we evaluated the Rankl/Opg (OPG, a negative regulator of osteoclastogenesis) gene expression ratio in the OCY-enriched bone fraction using RT-qPCR. We found no significant differences in the Rankl/Opg ratio between Doxo- and Veh-treated groups in the OCY-enriched bone fraction (Fig. S10A–C). While we failed to observe changes in OCYs, gene expression analysis of the bone-resident fraction from 12-week-old C57LB/6 mice treated with PTX showed a high Rankl/Opg ratio compared to Veh-treated samples (Fig. S10D–F), raising the possibility that senescent CAR and/or BMAd were the source of RANKL.

To ask if the senescent CAR/BMAds impacted the Rankl/Opg ratio, we returned to the INK-ATTAC transgenic mouse model (Fig. 6A). Immunohistochemistry staining and RT-qPCR results showed that the elimination of senescent CAR/BMAds by AP-treatment abolished Doxo-induced Rankl production and reduced the Rankl/Opg ratio in the bone-resident fraction (Fig. 6B–F). Accumulating evidence has revealed that marrow adipo-lineage cells including CAR cells and BMAds can express RANKL in pathological conditions of bone loss1,27,28, and our scRNA-Seq analysis suggested elevated expression levels of Rankl in Doxo-treated CAR cells compared to Veh-treated CAR cells (Fig. S10G), suggesting CAR cells were responsible for osteoclast differentiation. To confirm that senCARs and senBMAds were the source of increased RANKL in response to chemotherapy, we isolated floating senBMAds and sorted to enrich for senCARs (CD45-Ter119-CD31-Sca1-Pdgfrβ+) as previously described and found that the Rankl/Opg ratio was increased in cells from Doxo-treated versus Veh-treated mice (Fig. 6G–M), indicating that chemotherapy-induced senCARs and senBMAds support osteoclastogenesis through RANKL secretion.

To establish that it was BM adipo-lineage cell-derived RANKL that drove bone loss in response to chemotherapy, we first performed gene expression analysis of the Rankl/Opg ratio using the bone-resident fraction from 11-week-old ADQ-Cre+/DTR mice, we found no differences in cells from the Doxo- and Veh-treated groups (Fig. S10H–J). Thus, we next crossed the ADQ-CreERT2 mouse to Tnfsf11fl/fl mice to allow us to delete Rankl conditionally (referred to as ADQ-Cre/Rankl) in BM adipo-lineage cells (Fig. S10K). Prior to using these mice, we confirmed that Rankl was deleted upon Cre activation in femurs (Fig. S10L). To investigate whether BM adipo-lineage cell derived RANKL contributes to osteoclastogenesis in response to chemotherapy, we carried out TRAP staining on ADQ-Cre/Rankl mice and found reduced numbers of osteoclasts in Doxo-treated Rankl-/- mice compared with Doxo-treated control mice (Fig. S10M–O). Given this reduction in osteoclasts, we next asked how BV/TV was impacted in these mice. Thus, we harvested the femurs from Veh- and Doxo-treated 12-week-old female ADQ-Cre/Tnfsf11+/+ mice. Importantly, immunostaining revealed that p16+ cells remained detectable in Rankl-deficient mice, indicating that Rankl deletion does not prevent the accumulation of senescent cells (Fig. S10P, Q). μCT analyses revealed that deletion of Rankl from adiponectin positive cells prevented Doxo-induced bone loss as confirmed by unchanged trabecular bone volume (BV/TV), bone mineral density (BMD), trabecular number (Tb.N), and trabecular spacing (Tb.Sp) in Doxo-treated ADQ-Cre+/Rankl+/+ mice compared to Veh-treated mice (Fig. 6N, O, S11A–D). We also analyzed femurs from male mice and found the same results (Fig. S11E–I). Collectively, these findings indicate that RANKL derived from senCARs and senBMAds contributes to bone loss in both female and male mice.

Senolytics and senomorphics prevent chemotherapy-induced bone loss
Our use of the INK-ATTAC mouse model demonstrated that targeting senescent bone cells prevented chemotherapy-induced bone loss. To complement this work and explore the feasibility of pharmacologically targeting senescent bone resident cells in a therapeutic setting, we used a senolytic approach (5 mg/kg dasatinib and 50 mg/kg quercetin cocktail, D+Q) or senomorphics that target the p38MAPKα or MK2 kinase to target chemotherapy-induced senescent cells and SASP, respectively. To establish the specificity of the D+Q approach, we first examined the senescence profile in femur harvested from 12-week-old female Veh- and Doxo- or PTX ± (D+Q)-treated groups (Fig. 7A). As expected, we observed that D+Q significantly reduced Doxo- and PTX-induced SA-β-gal positive cells (Fig. S12A, B) as well as Doxo-induced EBF3+; SPiDER+ CAR cells (Fig. 7B, C). Next, to examine whether the elimination of senescent cells by D+Q could prevent chemotherapy-induced bone loss, we performed μCT and found that Doxo-induced bone loss was prevented by D+Q treatment (Fig. 7D, E). Other μCT bone parameters such as BMD, Tb.N, Tb.Th and Tb.Sp were also evaluated and altered as expected (Fig. S12C–F). In addition, the impact of PTX treatment was also tested and we found that D+Q also prevented PTX-induced bone loss (Fig. S12G–K). Furthermore, quantification of osteoclast number by TRAP staining showed a significant increase in osteoclast numbers (N.Oc/BS and Oc.S/BS) in Doxo-treated mice as expected that was prevented by D+Q treatment (Fig. 7F–H). Moreover, Doxo-induced reductions in OCN+ osteoblasts were also prevented by D+Q treatment (Fig. S12L, M), indicating that the pharmacologic elimination of senescent cells prevents the disruption of bone homeostasis.

Senescent cells produce a complex mixture of factors collectively termed the senescence-associated secretory phenotype (SASP) that is unique to the cell type undergoing senescence and the stress responsible for the induction of senescence29,30. The SASP is comprised of cytokines, chemokines, extracellular matrix proteases, growth factors, and other signaling molecules30,31. These factors can alter the tissue environment by activating cell surface receptors and their related signaling pathways, leading to several pathologies, including bone loss29,32. Previously, we showed that inhibition of the p38MAPK-MK2 pathway rescued chemotherapy-induced bone loss6. Here, we wanted to ask if bone resident senescent cells were impacted by p38MAPKα-MK2 inhibitors (p38i/MK2i). To address this, we performed immunofluorescence staining using a p16 antibody and found, as expected, that inhibition of p38MAPKα or MK2 (p38i or MK2i) failed to eliminate senescent cells in the bone (Fig. 7I, J). However, we found that within the bone marrow, the osteoclastogenic cytokine (RANKL), a component of the SASP33, was significantly reduced by both p38i and MK2i treatments (Fig. S13A, B). Moreover, RT-qPCR gene expression analysis of the bone-resident fraction showed decreased expression of additional SASP factors (e.g., Il6, MMP9, and Vegfα) in the Doxo+p38i/MK2i group compared to the Doxo-treated group (Fig. S13C), indicating that targeting the p38MAPKα-MK2 pathway can limit senescent cell-derived SASP factors. Subsequently, we asked if we prevented acute bone loss with p38i/MK2i. μCT analysis showed that inhibition of SASP by p38i/MK2i prevented chemotherapy (Doxo and PTX)-induced trabecular bone loss while no change in the cortical bones was observed (Fig. 7K, L, S13D–I. S13J–P). Collectively, these data demonstrate that pharmacological inhibition of the SASP prevents chemotherapy induced bone loss, raising the possibility that this could be a viable approach in the clinical setting.

Some studies have shown that senescent bone marrow stromal cells support osteoclast differentiation by secreting SASP factors such as RANKL that are closely linked to bone loss8,34,35. Therefore, next we evaluated osteoclast numbers by TRAP staining in 12-week-old female mice treated with Veh and Doxo ± p38i/MK2i. Results showed that Doxo significantly increases osteoclast numbers as expected that were reduced by p38i/MK2i (Fig. 7M–O). We also evaluated osteoblast-mediated bone formation rate over bone surface (BFR/BS) by calcein/alizarin red staining. As expected, bone formation was reduced in Doxo-treated compared to Veh-treated mice after 9 days while Doxo + p38i/MK2i groups restored bone formation activity similar to Veh-treated mice (Fig. 7P, Q). Taken together, these data demonstrate that chemotherapy-induced senescent BM adipo-lineage cells secrete SASP factors that in turn drive bone loss by directly altering the balanced action of osteoclasts and osteoblasts.

The use of the p38/MK2 inhibitors could affect all cells. Thus, we next wanted to confirm whether SASP derived from adiponectin-positive cells was sufficient to drive bone loss in response to chemotherapy. Thus, we mated the ADQ-CreERT2 mouse to a mouse that contained a floxed Mapkapk2 allele (referred to as MK2f/f herein) to specifically assess the role of MK2 in adiponectin expressing cells. ADQ-CreERT2/MK2f/f mice were treated with tamoxifen chow for 9 days and received Doxo or vehicle and bones were assessed nine days later (Fig. S14A). To assess the presence of senescent cells, we first stained bones for p16 expression and found that p16+ cells were high in the Doxo-treated group (Fig. S14B, C), indicating that loss of MK2 does not inhibit the induction of senescence. In contrast, when we assessed the bone, we found no significant differences in BV/TV between Veh- and Doxo-treated groups (Fig. S14D), indicating that MK2-dependent SASP derived from adiponectin positive cells drives acute bone loss in response to chemotherapy.

Senolytics prevent chemotherapy-induced bone loss in tumor bearing mice
Bone loss is a significant problem for breast cancer patients undergoing chemotherapy treatment and can lead to increased fracture risks and reduced overall survival36,37. Given the potent effects of senolytics on preventing chemotherapy-induced bone loss, we next wanted to ask how senolytics impacted the anti-tumor effects of chemotherapy. Using a clinically relevant mouse model, we asked whether targeting senescent cells by senolytics (D+Q) could rescue chemotherapy-induced bone loss within bones harboring metastatic breast cancer. For these studies, we utilized paclitaxel (PTX) because it is given as part of the neoadjuvant (or adjuvant) chemotherapy regimen for all breast cancer subtypes when chemotherapy is indicated. We injected 5 × 104 PyMT-BO-1 (GFP/Luc) cells intracardially into 12-week-old female albino C57BL/6J mice to broadly distribute metastatic cells. Mice were then randomized to treatment groups that consisted of Veh, PTX (50 mg/kg), or PTX + (D+Q). Mice were sacrificed on day 13 when Veh treated mice became moribund (Fig. 8A). We first assessed senescence induction in this model by staining femurs with the SPiDER-β-gal probe and found that D+Q treatment decreased PTX-induced SA-β-gal+ cells (Fig. 8B, C). Co-staining for EBF3 and SPiDER revealed D+Q reduced senCARs cells (EBF3+; SPiDER+) in mouse femurs (Fig. 8D, E). Importantly, bioluminescence imaging of metastatic tumor cells revealed that D+Q treatment did not impact the anti-tumor effects of PTX (Fig. 8F, G). Consistently, H&E staining of tumor-bearing bone tissues further confirmed these findings (Fig. S15A). However, while μCT analysis of the femurs showed significant bone loss in PTX-treated mice, the addition of D+Q prevented trabecular bone loss (Fig. 8H, I) without affecting cortical thickness (S15B, C). Osteoclast numbers were greatly reduced when PTX treated mice also received D+Q (Fig. 8J–L). However, PTX plus D+Q did not lower tumor burden compared to PTX alone in TRAP-stained samples, consistent with BLI and H&E staining results (Fig. 8J, S15D). Whereas osteoblasts (OCN+ cells) were significantly increased when mice were treated with PTX and D+Q compared to PTX alone (Fig. S15E, F). Together, these results demonstrate that targeting senCARs and senBMAds to prevent chemotherapy-induced bone loss during cancer treatment preserves the anti-tumor effects of chemotherapy.

Methods
Animals and treatment
All animal experiments were performed in compliance with Washington University in St. Louis’s Animals Studies Committee. All animal procedures were approved by Washington University’s Institutional Animal Care and Use Committees (IACUC). Mice were maintained on a 12-hour light/12-hour dark cycle, with ambient temperature between 20–22.2 °C and relative humidity of 30–70%. Mice were maintained on a regular chow diet (PicoLab Rodent Diet 20, Cat. No. 5053) formulated with 20% protein. C57BL/6J (JAX, #000664) and albino C57BL/6J mice (JAX, #000058) mice were purchased from the Jackson Laboratory. The INK-ATTAC and p16-CreERT2/tdTomato mouse models were generated by Darren Baker’s group10 and Makoto Nakanishi’s group19, respectively. ADQ-Cre/DTR and ADQ-Cre/QR mice were generated by crossing C57BI/6 ADQ-Cre transgenic mice with DTRf/f and INK-ATTACf/f (INK-QR) mice, respectively. AdipoCreERT2/Rankl and AdipoCreERT2/MK2 mice were generated to delete the gene of interest conditionally by crossing C57BI/6 ADQ-CreERT2 transgenic mice with Ranklf/f and MK2f/f mice, respectively. Mice harboring the AdipoCreERT2 transgene were fed tamoxifen chow to induce Cre recombinase activity. In this study, both female and male animals were included in the experiments unless otherwise specified. For tumor studies, a metastatic tumor model was established using PyMT-BO-1 (GFP/Luc) cells in albino mice. Briefly, 50 μl of a single-cell suspension containing 50,000 PyMT-BO-1 (GFP/Luc) cells was injected directly into the left cardiac ventricle (intracardiac injection) under anesthesia. On day 2 post-injection, metastatic lesion formation was evaluated and confirmed by in vivo bioluminescence imaging (BLI). Mice were then randomized into different treatment groups. Paclitaxel (PTX, 50 mg/kg) was administered intravenously (I.V.) on days 4 and 5, followed by a combination of dasatinib and quercetin (D+Q) administered every other day for a total of four doses. On day 13, both in vivo and ex vivo BLI were performed to assess metastatic tumor burden, and mice were subsequently sacrificed for tissue collection and further analyses. Experimental endpoints were determined by clinical signs associated with metastatic burden, including weight loss exceeding 20% of body weight. All mice were maintained in groups of three to five animals per cage with water and food ad libitum. Mice were euthanized using CO2. The intact femurs and tibias were removed for further analysis. Doxorubicin (LC Laboratories) was prepared in water and administered in a single dose at 5 mg/kg via intraperitoneal (i.p.) injection, and paclitaxel (TOCRIS, a Biotechne brand) was administered in two doses at 50 mg/kg via tail vein (IV). AP20187 (Batch No.: A113-20, Chemvada Life Sciences, San Diego, CA) and prepared with 100% ethanol: polyethylene glycol 400: 2% Tween-20 in molecular water at 4:10:86. AP20187 was administered for a week at 10 mg/kg thrice through i.p. injections. Mice used in this study were genotyped by TransnetYX (an automated genotyping company, USA). Mouse details including strain, source/catalog number and injected compounds are provided in Supplementary Table 1.

Bone marrow transplantation
Bone marrow transplantation was performed according to previously described protocols5. Recipient C57BL/6 CD45.1 mice received two doses of 400 cGy 4 hours apart, followed by transplant of bone marrow by retro-orbital injection (i.v.). Irradiation was carried out using an X-ray irradiator (XRAD320). Donor bone marrow was prepared from INK-ATTAC CD45.2 mice as follows: donor mice were sacrificed by CO2 inhalation, both femurs, tibias and ilia were extracted in a sterile setting and flushed using pulsed centrifugation to collect marrow. Bone marrow was reconstituted in cold sterile serum-free 1X HBSS and injected retro-orbital at a concentration of 5 million cells per 200 μl per mouse. Mice were monitored over 2 weeks for signs of radiation sickness. 6-week post irradiation, mice were given paclitaxel and sacrificed 9 days later.

Vertebral body transplant (vossicle) implantation
Vertebral bodies were harvested from 4-day-old INK-ATTAC pups. Soft tissues were stripped, and the vertebrae were sectioned into single vertebral bodies (Lumbar 4 and 5) with a scalpel blade. Female 11-week-old C57BL/6J mice used as transplant recipients were anesthetized with isoflurane inhalation. Two 1-cm subcutaneous incisions and pouches were made at both sides of the flank region of each mouse. L4 and L5 were washed with PBS and implanted in the right and left pouches of each mouse, respectively. The surgical sites were closed using a surgical stapler.

μCT scanning and assessment of the femur
Femurs were harvested from euthanized mice and after stripping the skin, the femurs were fixed with 10% neutral buffered formalin (NBF) overnight at 4°C with gentle rotation. The bones were washed in phosphate buffered saline (PBS) and stored in 70% ethanol to be used for μCT. For μCT analysis, bones were suspended in 2% agarose and scanned using microcomputed tomography (μCT 50, Scanco Medical)) at 70 kVp, 114 μA, and 20 μm resolution. For the trabecular compartment, contours were traced on the inside of the cortical shell using 2D images of the femoral metaphysis. The end of the growth plate region was used as a landmark to establish a consistent location for starting analysis, and the next 100 slices were analyzed. The following trabecular parameters are reported for all μCT experiments: bone volume over total volume (BV/TV)), bone mineral density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). For the cortical compartment, contours were traced on the outside of the cortical shell using 2D images of the femoral mid-diaphysis and 50 slices were analyzed. 3D reconstructions right below the growth plate of the femur were generated using Scanco μCT 50 Ray Tracer software.

Bone histomorphology and tartrate-resistant acidic phosphatase (TRAP) staining
At sacrifice, mice femurs were isolated and fixed in 10% NBF for overnight. Bones were decalcified in 14% EDTA (Sigma, E5134), pH 7.2 for 14 days and embedded in paraffin. 5 μm sections of proximal metaphysis were prepared using a microtome (Leica, RM2235). Standard H&E and TRAP staining techniques were used for all bone sections. Images were collected using the Zeiss Axio Scan Z1 Brightfield slide scanner. To quantify the TRAP-positive cells and determine how much of their surface area covered the bone, we used the BioQuant 2 software to quantify osteoclasts per bone surface (Oc.S/BS and N.Oc/BS). We carried out these measurements by defining a region of interest with the stamp tool window. We then chose a diagonal line type and set the distance to the growth plate and measured the N.Oc/BS and Oc.S/BS.

Double bone labeling
Mice were administered calcein (10 mg/kg, Sigma #C0875) and alizarin red (30 mg/kg, Sigma #A3882) via i.p. injection 9 and 2 days prior to sacrifice, respectively. Femurs were fixed with 70% ethanol overnight at 4°C with gentle rotation, then embedded in methylmethacrylate (MMA) for sectioning. Sections were left unstained and images were collected using the Zeiss Axio Scan Z1 fluorescence slide scanner, WUCCI. Histomorphometric analysis was performed using BioQuant software to quantify bone formation rate per bone surface (BFR/BS).

Single-cell RNA-sequencing
12-week-old mice were treated with Veh- or Doxo (10 mg/kg) and sacrificed at 96 hours. Bones (femurs and tibias) were harvested from euthanized mice and placed in cold DMEM/F12 (Gibco). Muscle and connective tissues were removed. Bones were gently ground and then cut into small fragments. Fragments were transferred and digested with fresh 2 mg/ml collagenase (Sigma, C0130) in DMEM/F12 on a rotating water bath at 37°C for 30 min. Following the first digestion, the released cell suspension was filtered through a 70-μm nylon mesh into a collection tube and placed on ice. A second digestion of the remaining fragments was performed and the cells suspension was filtered into the same collection tube. Reactions were quenched with FACS buffer (PBS plus 0.5% BSA) with 2 mM EDTA. Following FACS buffer wash and RBC lysis, dissociated cells were labeled with anti-CD45 magnet microbeads (Miltenyi Biotech) and enriched into a CD45+ fraction and CD45- fraction by MACS (magnetic-activated cell sorting, Miltenyi Biotech). Cells were stained with fluorochrome antibodies for 20 min on ice for cell sorting. We used CD45 (BioLegend, 103126), CD71 and Ter119 to deplete CD45+ CD71+, Ter119+ populations using a BD FACSAria III Cell Sorter. After sorting, cells were delivered for library construction using 10X Genomics Chromium 3′ GEM Single-Cell Library v3 kit (10X Genomics). Sequencing was performed according to standard pipeline at The Genome Technology Access Center at Washington University, St. Louis. The Cell Ranger Software Suite from 10X Genomics was used for sample demultiplexing, barcode processing, and single-cell counting. The Cell Ranger count was used to align samples to the reference genome GRCm38 (mm10). For data analysis, the filtered feature barcode matrices were imported into Rstudio and analyzed using Seurat55.

Each Seurat object was filtered to exclude genes expressed by less than 3 cells, cells expressing less than 200 genes or more than 10,000 genes, cells with more than 10% mitochondrial RNA content and cells with less than 100 counts or more than 100,000 counts. Log-based normalization, identification of variable features and scaling was performed using the corresponding Seurat functions (NormalizeData, FindVariableFeatures and ScaleData). Next, principal component analysis (PCA) and imputation using adaptively threshold low-rank approximation (ALRA)56 was performed on the object. UMAP dimensional reduction was performed using the first 35 dimensions. We then used the FindNeighbors and FindClusters functions to cluster cells at a resolution of 0.1. Canonical markers were used to identify immune (Ptprc+) and endothelial clusters (Epcam+, Cdh5+). Bone cells that were not immune or endothelial cells were re-clustered and canonical markers were used to identify the different bone cells. Due to the small size of the pericyte cluster, it was excluded from the final object named “Bone cells”. The final object was then carefully inspected for markers of senescence. DGE analyses between clusters were performed using MAST analysis57 and the obtained gene list was pre-ranked for gene set enrichment analysis (GSEA) based on p-values and log fold change. All visualizations of gene expression were performed using either the RNA or the ALRA assay of the dataset and are indicated accordingly in the figure legends.

For the analysis of the publicly available human bone scRNA-seq dataset (GEO: GSE230295) from acute lymphoblastic leukemia patients18, we utilized N=4 patient samples, with peripheral blood samples excluded. As formatted in the original dataset, samples collected at diagnosis (day 0) and post-treatment (day 15) were pooled for each patient prior to analysis. A merged object comprising all four patient samples was generated, and CD45+ hematopoietic cells were excluded by sub-setting cells with detectable Ptprc expression. The remaining CD45- stromal populations were normalized and reclustered using 10 principal components and a resolution of 0.2. All downstream analyses and plots were performed using this CD45- object.

Reverse transcription qPCR
To quantify gene expression, the marrow cavity was flushed out by centrifugation at high speed for 10 s at 4°C. Bones devoid of marrow were lysed and homogenized in TRIzol (Invitrogen). RINO Bullet-Blender Navy Bead Lysis tubes and Bullet Blender® Homogenizer were used for crushing the bone thrice at 4°C with high speed for 5 min. Once the bones were completely crushed, the tubes were centrifuged at 9000 xg for 10 min at 4°C. The supernatant was transferred to a new tube for RNA extraction. Total RNA was extracted using the RiboPure™ RNA Purification Kit (AM1924, Invitrogen). cDNA was prepared from RNA (1 μg) using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) followed by preamplification. RT-qPCR was performed using the Taqman PrimeTime® Gene Expression Master Mix kit and gene-specific primers and probes. All reactions were performed in duplicate. mRNA was normalized to the mean of two housekeeping genes (either cyclophilin with actin or TBP with tubulin). All primer sequences used in this study are listed in Supplementary Table 1.

Senescence associated β-galactosidase staining
SA-β-gal staining in femurs of mice was performed on frozen sections that were fixed in 10% NBF overnight at 4°C with gentle rotation. Next day, following PBS wash, decalcified in 14% EDTA (pH 7.2) for 3 days at 4°C with gentle rotation. Then, placed in a solution of 30% sucrose overnight. Next day embedded in OCT compound (Fisher Health Care, 4585) and sectioned 10 μm using cryotome (Leica, CM1950). Slides were washed in PBS for 2 min and then submerged in X-gal solution (1 mg/ml 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, 150 mM NaCl, 2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 40 mM NaPi pH 6.0, in H2O). The X-gal solution was passed through a 0.22 μm filter prior to use in order to remove particulate. Slides were kept at 37°C in the dark until the stain developed (∼6 h). After staining, slides were washed in PBS, and then the nuclei were counterstained using Nuclear Fast Red (Sigma). Slides were scanned by Zeiss slide scanner and SA-β-gal positive area was analyzed by HALO software.

Isolation of osteocyte-enriched cells
Briefly, mouse femurs and tibias were harvested and stripped of soft tissues followed by crushing. Subsequently, digested with fresh 2 mg/ml collagenase twice at 37°C for 30 min. The remaining cell fraction represents a highly enriched population of osteocytes8, which was used for gene expression analysis.

Immunofluorescence staining (frozen sections)
Harvested bones were fixed in 10% NBF overnight and decalcified in 10% EDTA for 3 days at 4°C followed by dehydration with 30% sucrose. Bones were embedded in OCT compound and femurs were cut longitudinal at 10 μm using a cryotome. Sections were washed, permeabilized for 10 min in 0.2% Triton-X in PBS and blocked for 1 h at room temperature using 0.5% BSA (Sigma, D9663) with Fc blocker (1:200) prior to incubation for overnight at 4°C with rabbit anti–p16 antibody (1:100, Cat. No. PA1-46220, Invitrogen) and mouse anti-PPARγ (1:300, Cat. No. 66936-1, Proteintech). The specificity of the p16 antibody was evaluated prior to use in p16-knockout mice femur (Fig. S2I). Next day, sections were washed with TNT buffer (0.1 M Tris–HCL pH 7.4, 0.15 M sodium chloride, 0.1% Tween-20) followed by incubation with secondary goat anti–rabbit Alexa Fluor 568 antibody (1:400, Cat. No. A-11011, Invitrogen) and goat anti–mouse Alexa Fluor 488 antibody (1:500, Cat. No. A-11001, Invitrogen). All sections were stained and mounted using SlowFade Gold antifade reagent with DAPI (Invitrogen S36939) according to the manufacturer's instructions. Mouse on mouse blocking was performed when anti-mouse antibody was used according to manufacturer instruction (Vector Lab. PK-2200). All fluorescent images were captured and scanned using a Nikon Eclipse 90i microscope or Zeiss Axio scan Z1 fluorescence slide scanner. Osteocalcin+ (OCN+) osteoblasts were analyzed using HALO software, with specific attention given to accurately quantifying bone surface-associated OCN+ cells. To ensure the precision of our analysis, OCN+ signals located within the marrow cavity were carefully excluded. However, OCN+ signals in close proximity to the bone surface were included. Importantly, the same criteria and analysis parameters were uniformly applied across all treated and untreated groups to ensure consistency and comparability. Antibodies details including catalog number and identifiers are listed in Supplementary Table 1.

Isolation of bone marrow adipocytes
Mature adipocytes (BMAds) were isolated directly from the bone marrow of mice according to previously described protocols21,22. Briefly, femurs were collected from mice, and the two ends of the bones were snipped. The bones were placed in a small microcentrifuge tube (0.5 ml) that was cut open at the bottom. The small tube with the bones was then placed into a bigger microcentrifuge tube (1.5 ml). Fresh bone marrow was spun out by quick centrifuge and resuspended in PBS. After centrifugation (900 xg, 5 min, RT), floating adipocytes were collected from the top layer and washed with PBS for 3 times.

In Vitro differentiation of bone marrow cells
BMSCs were isolated from the femurs and tibias of 12-week-old wild-type mice treated with Veh or Doxo. After 9 days of treatment, the femurs and tibias were harvested and the proximal ends of the bones were cut followed by isolation of BM with the aid of a syringe. To check the differentiation potential of BMSC, we cultured isolated BMSCs directly in osteogenic or adipogenic induction media. For osteogenic induction, BMSC were cultured in 12-well plates at a density of 1×104 cells/well. The medium was changed every 3 days. After culture for 14 days, the medium was removed, and cells were washed twice with PBS, then alkaline phosphatase (ALP) staining was performed where alkaline phosphatase substrate BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) (Sigma Fast, B5655) was added and incubated for 30 min at room temperature (RT). For alizarin red staining, after culture for 21 days, the medium was removed, and cells were washed twice with PBS, then fixed with 4% paraformaldehyde at RT for 30 min, after which they were rinsed twice with PBS. Finally, cells were stained with alizarin red S dye (Sigma, A5533) for 30 min and then rinsed thrice with distilled water for the removal of unbound stain, after which stained calcium nodules were captured by inverted microscopy. For adipogenic induction, BMSC were seeded in 12-well plates at a density of 1 × 104 cells/well and at day 14 images were captured. For lipid droplet staining, BMSC were seeded on coverslips (22x50 mm) at a density of 1 × 104 cells and at day 14, cells were washed twice with PBS and fixed with 4% paraformaldehyde at RT for 30 min. They were then washed twice with PBS and stained with 1µg/ml of lipophilic fluorochrome BODIPY 493/503 (Invitrogen, D3922) for 15 min at RT. They were then washed thrice with PBS, mounted using SlowFade Gold antifade reagent with DAPI and stained lipid droplets were scanned using the Zeiss Axio Scan Z1 fluorescence slide scanner, WUCCI.

Oil Red O Staining
Frozen sections were allowed to air dry at room temperature for 1 h and washed with water for 5 min followed by 60% isopropanol wash for 15 sec. Oil red O (Sigma, 00625) prepared according to the supplier was added and incubated for 15 min with gentle shaking in the dark. After incubation, rinsed with 60% isopropanol for 15 sec followed by hematoxylin staining and then, mounted using permanent mounting medium. For SA-β-gal/Oil red O-double staining, frozen sections were first immersed in SA-β-gal solution and incubated for 5–6 h at 37°C. After that, slides were washed and stained with oil red O staining. Oil red O-stained positive cells or SA-β-gal/oil red O-double positive cells were visualized and captured using an inverted microscope (Nikon Eclipse 50i) equipped with a Nikon DS-Fi3 camera with 20x magnification. SA-β-gal/oil red O-double positive area were analyzed by HALO software.

Oral feeding of p38i/MK2i compounded chow
The p38MAPK small-molecule inhibitors CDD111 (p38i) and CDD2231 (MK2i) (Aclaris Therapeutics, Inc.) were compounded at 1000 PPM. Both were compounded into Research Diets Inc., catalog number 5001. Female C57BL/6 mice were fed ad libitum. Mice were randomized onto inhibitor-containing or regular chow on the same day as their first doxorubicin dose till 9 days.

IC injection and BLI
12-week-old female albino C57BL/6J mice were anesthetized with 5 μl/g body weight of Ketamine/xylazine cocktail (17.7 g/ml of ketamine and 2.65 mg/ml of xylazine). When animals were completely anesthetized, 50 μl of PyMT-BO-1 (GFP/Luc) cells (50,000 cells) were injected directly into the left cardiac ventricle (IC injection). The PyMT-BO-1 cell line was gifted by Kathy Weilbaecher. Prior to use, we verified the PyMT-BO-1 cells using STR profiling (ATCC) and mycoplasma testing. Bioluminescence imaging (BLI) was carried out as previously described58. Ex-vivo imaging was performed on an IVIS100 or IVIS Lumina (PerkinElmer; Living Image 3.2, 1–60 sec exposures, binning 4, 8, or 16, FOV 15 cm, f/stop 1, open filter). Mice were injected with D-luciferin (150 mg/kg in PBS; Gold Biotechnology) via i.p. After being sacrificed, both hind limbs were harvested and imaged for 10 seconds. For analysis, total photon flux (photons/sec) was measured from a fixed region of interest (ROI) over the whole bones using Living Image 2.6 software.

Statistics
All statistical analyses were carried out using Prism (latest version 10.4.0). Data are expressed as mean ± SEM. Between two group comparisons, unpaired two-tailed Student t-tests were used. For multiple comparisons, one-way ANOVA with Tukey test was used. A p-value of less than 0.05 was considered statistically significant. Quantitative assessments of μCT-based analyses were performed by an individual who was blinded to the sample identity.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Manuscript summary\nThis study provided mechanistic insights into how chemotherapy-induced senescence leads to bone loss. By using scRNA-\nseq and performing validation experiments on multiple genetically modified mice in non-tumor setting, the authors provided\ncompelling results that chemotherapy-induced senescence is restricted to bone marrow adipo-lineage cells, and these cells\ncontributed to bone loss through increased expression of Rank ligand, which led to increases in osteoclast number and\nactivity. The authors also evaluated the effect of senolytic drugs in protecting against bone loss in tumor-setting (breast\ncancer), though further investigation is needed in the future. Overall, this work presented a novel finding that targeting\nsenCAR/senBMAds may be a promising approach to prevent bone loss caused by chemotherapy. There are many strengths\nto this report, including complete and rigorous experiments and data analysis in vitro and in vivo. The findings have high\nclinical relevance. These data provide mechanistic insight. However, there are some limitations, detailed below, which\nshould be addressed.\nQuestions\n1. Can chemotherapy cause direct senescence in osteoclasts (OCs)?\nFig.1F shows increased expression of p16 in crushed bone devoid of bone marrow cells, which indicates that senescence\noccurred in cells on or in the bone. Fig 1J shows decreased OCs in doxorubicin (Doxo) group treated with AP20187 (AP, the\ndrug that eliminates p16+ senescent cells). Considering AP eliminates p16+ cells systemically, can the decrease in OCs be\ndue to the elimination of AP on senescent OCs?\n2. The statement on “systemic senescent factors do not contribute to bone loss” (line 183-184) is overstated.\nIn lines 180-184, the author stated that “AP did not rescue bone loss and reduce TRAP+ OCs in WT, indicating that systemic\nsenescence factors do not contribute to bone loss”. By using vossicle model (Figure 2), implanting vertebrate from INK-\nATTAC mice in C57BL/6 WT mice, the observation that AP protected bone in INK-ATTAC vertebrate but failed to prevent\nbone loss in WT femur can only indicate that bone resident cells contribute to Doxo-induced bone loss. In order to assess\nthe effect of systemic factors, reversed implantation (implanting vertebrate from WT mice in INK-ATTAC mice) will need to be\nperformed. If similar bone loss was observed in WT vertebrates implanted in INK-ATTAC mice with Doxo and AP treatment\ncompared to the group with Doxo only, it may be indicated that systemic senescence factors do not contribute to chemo-\ninduced bone loss.\n3. Clarification is needed for what type of sample is used for scRNA-seq.\nLine 212 states that the author performed scRNA-seq on bone marrow. However, in Fig. 3A, a step in the schematic shows\nbone crushing, which causes confusion about whether whole bone (crushed bone + bone marrow cells) or only bone\nmarrow cells should be used for scRNAseq. Based on the context, it is likely that the author used bone marrow cells.\nHowever, considering that senescence can occur in bone resident cells (see question 1), sequencing only on bone marrow\ncells rules out the possibility of senescent bone resident cells contributing to chemotherapy-induced bone loss.\n4. Related to Figure 4\na. In lines 251-252, it states that “no significant changes in p16+/pparγ- cells across the groups”. There are no images in Fig.\n4 that support this statement.\nb. In line 271, it states that “there were no changes in Ebf3-SPiDER+ cells in Veh and Doxo groups”. What about Ebf3-\n;tdTom+ cells?\nc. Fig. 4M needs a clear label. Based on the results description, these µCT images were taken from DTRADQ mouse treated\nwith PBS or DT. PBS should be labeled above the first paired µCT images, and DT instead of iDTRADQ should be labeled\nabove the second paired µCT images.\n5. Related to Figure 5\nIn line 339 and Fig. 5C-F, please clarify which bone samples were used for RT-qPCR, bone marrow, crushed bone\n(depleted bone marrow), or whole bone?\n6. Related to Figure 6\na. Fig. 6M, IF staining of RANKL. Can the author include a description of the location of the stained RANKL in text? Is it\nprimarily present in the bone marrow space or on the bone?\nb. Line 412-413 states that the expression of other SNAP factors decreased after p38i/MK2i in crushed bone depleted bone\nmarrow. Since senCAR/BMAds reside in bone marrow, expression of these SNAPs factors needs to be detected in bone\nmarrow to further confirm the inhibitory effect of p38i/MK2i on SNAPs.\n7. Related to Figure 7\na. Representative images are needed for Fig. 7B\nb. For Fig.7E-F. Bioluminescent images and intensity quantification are not sufficient to assess tumor burden. Please show\nhistological images of tumor-bearing bones and quantify tumor burden by histomorphometry. Histomorphometric analysis is\nthe gold standard for measuring tumor burden in bone.\nc. Fig. 7G. How to distinguish bone loss caused by PyMT-BO-1 cells from paclitaxel (PTX)?\nd. In Fig. 7J, tumors are not observed on the histological section, which is inconsistent with the bioluminescent images\n(showing tumor grew in bones). High-resolution images need to be provided in order to show tumor.\ne. Given that (1) PTX and Doxo may have different anti-tumor effects on breast cancer, and (2) senolytic drugs (D+Q) may\ninteract differently with chemotherapies, please provide justification for why PTX, not Doxo, was chosen to use in the breast\ncancer bone metastatic model.\nOverall, the data presented in figure 7 is not of the same quality as previous figures. The rationale for using the bone\nmetastatic model to support the authors’ findings is not clear. By introducing tumors in the model, it complicates the results\nand makes it hard to evaluate the changes in bone due to tumors or chemotherapy. The author may consider excluding the\nfigure 7 from the manuscript since the model used here is not appropriate, and the results do not align with or support the\nmain conclusion. Future work could investigate the author’s findings by using adjuvant breast cancer models.\n8. Others\na. Detailed methods are needed on how the author defines and measures the OCN+ area on bone sections.\nb. In line 271, typo. Fig. 4SL should be Fig. S4L\nc. The phrase “bone resident cells” is used multiple times throughout the manuscript. The authors seem to use it to refer both\nto bone marrow cells and cells in/on bone, which causes confusion. Please clarify it in text or figure legend, as appropriate.", "reviewer_2": "Evaluation report for the manuscript entitled “Chemotherapy-induced adipo-lineage cell senescence drives bone loss” by\nGK Raut and collaborators submitted to Nature Communications.\nGK Raut et al. previously reported that elimination of senescent cells and inhibition of SASP production using genetic and\npharmacological approaches inhibiting the p38MAPK-MK2 pathway prevented chemotherapy-induced bone loss. In this\nreport, by using an extensive number of mouse models, genetic and pharmacological approaches, the authors propose that\nchemotherapy-induced senescence does not apply to osteocytes, as previously reported by Sundeep Khosla’s group\n(PMID: 27341653). Instead, the authors found that chemotherapy-induced senescence is limited to bone resident CAR cells\n(senCARs) and, also, bone marrow adipocytes (senBMADs), which crucially contribute to bone loss through RANKL ligand-\ndriven osteoclast activation. In addition, chemotherapy-induced senescence reduces osteoblast differentiation and the bone\nmineralization capacity resulting in a decoupling process of bone formation and resorption. Interestingly, the authors provide\nevidence that genetic and/or pharmacological (dasatinib + quercetin) elimination of senCARs and SenBMAds ameliorated\nchemotherapy-induced bone loss without affecting the anti-tumour activities of chemotherapy. Moreover, senomorphic\ntargeting of RANKL (described as a SASP factor) by using p38i and MK2i inhibitors prevented chemotherapy-induced bone\nloss. Altogether, GK Raut and collaborators propose a therapeutic strategy to preserve bone integrity and improve quality of\nlife during chemotherapeutic cancer treatments.\nThis is a well-presented and voluminous article in a hot topic of potential clinical relevance. The experiments are solid and\nelegantly designed, and the results align with the interest for a broad audience. Overall, the conclusions seem supported by\nthe experimental data, however the manuscript must address the conceptual advance of this research, including conflicting\ndata with other groups, and provide further experiments to reinforce the conclusions before it can be recommended for\npublication in Nature Communications.\nMain points\n1. The authors must address the conceptual advance of this work for a journal like Nat Comm, as they previously reported\nthat chemotherapy-induced senescence drives bone loss using the same models. i.e. doxorubicin- and PTX-mediated, INK-\nATTAC mice, and showing that clearance of senescent cells rescues osteoblast function and reduces osteoclasts numbers\n(Yao et al. Cancer Research 2020, PMID: 31932453). Also, in the same previous publication, the authors showed that the\nprocess of bone loss is driven by the p38MAPK-MK2 pathway, and senomorphic treatment with p38i/MK2i inhibitors limits\nSASP production and rescues chemotherapy-induced bone loss. Therefore, the novelty of the current study seems\ncompromised and mainly stems from the identification of senCARs and SenBMAds as key players of the observed\nphenotype. In addition, it is well known that osteocyte RANKL is required for cortical bone loss with age and that it can be\ninduced by senescence (Kim et al. JCI insight 2020, PMID: 32870816).\n2. It seems concerning a recent publication from another laboratory (Ali et al. Scientific Reports 2025, PMID: 40389522) reporting that the adverse effects of chemotherapy on bone mass are not prevented by senolytics. In particular, because\nthese authors use similar chemotherapies (i.e. doxorubicin, docetaxel and cyclophosphamide) and senolytics (i.e. dasatinib\n+ quercetin and piperlongumine). Also, the readouts are fundamentally the same, like trabecular bone mass, thickness, etc.\nThis point needs to be convincingly addressed. How do the authors explain this apparent discrepancy?\n3. Following with the previous point, a possibility raised by Ali and collaborators is that the decrease in osteoblast numbers\nis due to chemotherapy DDR-induced osteoblast apoptosis. Therefore, mechanistic analyses in this manuscript must\ninclude a comprehensive characterisation of apoptosis (e.g. TUNEL or activated-caspase 3) damage makers (e.g. gH2AX)\nand how they relate to the crucial cellular types that maintain the intricate balance of bone damage/loss and\nformation/resorption.\n4. The authors use the well-established INK-ATTAC mouse model to provide evidence that chemotherapy-induced\nsenescence associates with disrupted bone homeostasis, and also p16CreERT2-tdTom mice to identify CARs and BMADs\nwith p16 “senescent” features. To ensure specificity of the p16 antibody the authors tested bones from p16KO treated with\nDoxorubicin. If the authors are correct in their hypothesis, I find crucial to validate the main conclusions of this manuscript in\nchemotherapy-treated p16KO mice. What would be the phenotype and mechanism of this model in bone loss and\nsenescence induction?\n5. Also, the authors do not validate the potential role of p21 as senescence biomarker over the manuscript. In fact, the\np53/p21 axis is an essential component of the chemotherapy-induced DDR response. Are senCARs and senBMAds\npositive for both p16 and p21? Or there are different senescent ecosystems? In this regard, district secretomes in p16 and\np21 positive senescent cells have been proposed across tissues, including the bone (Saul et al. bioRxiv,\nhttps://www.biorxiv.org/content/10.1101/2023.12.05.569858v1.full.pdf).\n6. Any validation with human datasets / samples post-chemotherapy regarding senCARs / senBMAds would reinforce the\nconclusions and translatability of this study, although I’m aware of the difficulties in getting such samples.\nOther points\n- The Discussion is currently underdeveloped and should address all concerns regarding novelty, contradictory data with\nother groups, and the limitations of this study.\n- RNAseq analyses rely on SenMayo datasets, but it would be important to correlate the data with other datasets and\nsenescent signatures.", "reviewer_3": "In the outlined manuscript, Ganesh Kumar Raut and colleagues describe in a compendium of in vivo experiments using\nseveral genetic mouse lines, that chemotherapy-induced bone loss is linked to the induction of senescence specifically in\nadipo-lineage cells of the bone. Adipo-lineage cells include CXCL12-abundant reticular (CAR) cells and mature adipocytes,\nboth marked the expression of adiponectin in mouse bones. In previous findings from the group (Yao et al., doi:\n10.1158/0008-5472.CAN-19-2348), they showed that chemotherapy induced bone loss can be prevented by the ablation of\nsenescent cells using a Cdkn2a driven suicide gene (INK-ATTAC) or the use of senormorphics. In the previous publication\nthey used a 28-day exposure to Doxo or PTX, showing that trabecular bone loss is due to loss of trabecular numbers and\nthickness, that aligns with an increase in osteoclast numbers and decreased bone formation, all of which was reversible\nthrough ablation of senescent cells.\nIn the current study the group expands these observations by the identifying the putative cell type, i.e., cells of the adipo-\nlineage, being susceptible to the induction of senescence which in turn drives the expression of RANKL and consequently\nosteoclast mediated bone loss. However, the authors do not provide regulatory mechanisms, i.e., which signals, and\npathways confer the high susceptibility of CAR cells and adipocytes to chemotherapy treatment, and whether those signals\ndirectly or the SASP gene signatures drives expression of TNFSF11 specifically in adipo-lineage cells. In addition, the\nauthors do not provide information whether baseline adipocyte content, i.e., marrow adiposity due to ageing, obesity, or\ncaloric restriction, has an impact on the degree of chemotherapy induced bone loss. Taken together, these new insights are\nlimited in the scope of Nature Communications and its broad audience.\nWhile depletion of adiponectin expressing cells or TNFSF11(RANKL) ablation specific to adiponectin expressing cells\nconvincingly shows to prevent chemotherapy induced bone loss, the evidence of the senescence signature being restricted\nto CAR cells and adipocytes is less supported. The latter is mainly due to the usage of a variety of genetic mouse models or\nstaining procedures which are not directly comparable (often using several methods to stratify the adipo-lineage but using a\nsingle approach to deny the role of other bone resident cells). This is surprising as the authors performed experiments, such\nas the adipocyte fractionation and scRNA-sequencing in which different compartments could have been compared using\nthe same method to underline the susceptibility of adipocyte-lineage cells to senescence.\nIn addition to that, the phenotype of trabecular bone loss characterized in supplementary figure 1 is barley recapitulated\nthroughout the intervention studies and often bone loss monitored through loss of BV/TV does not correspond to changes in\ntrabecular numbers or thickness, questioning the cause of bone loss.\nMajor points:\nTrabecular bone loss has been initially characterized by loss of trabecular numbers, however this parameter is not changed\nthroughout the intervention studies in the INK-ATTAC mice, adipocyte depleted mice, mice lacking RANKL in adipocytes, or\nmice treated with senolytics or senomorphics. In addition, the effects of trabecular thickness are limited and therefore are\nunlikely to explain the observed bone loss. Did the authors check mineral content and density to explain the loss of\ntrabecular bone without effects on trabecular numbers and thickness.\nThe authors use a lot of space for the clustering the cell-type identification in the scRNA-seq data, but do not follow up on\nthis throughout the manuscript. Therefore clustering could be part of the supplement. In contrast, the gene set enrichment\nanalysis should be performed by comparing Doxo condition to vehicle for each individual cluster to highlight that CAR cells\nare more susceptible to the induction of senescence than the other cells. For now, the authors compare CAR cells with the\nremaining cells, but not the induction levels in each cluster individually. A heatmap of the Sen-Mayo gene signatures could\nbe additionally used to quantify changes in senescence associated gene transcription.\nAlong the line, the fractionation of mature adipocytes for gene expression (Fig. 4D) should be complemented by analysis of\nthe SVF fraction, i.e., the cells in the pellet, to compare induction levels of SASP factors across fractions.\nWhile the authors emphasize to outline adipo-lineage cells to be highly susceptible to chemotherapy induced senescence\nand causally involved in bone loss, they do not show how adipocyte ablation and TNFSF11 conditional knockout are related\nto the senescence burden within the bone. Thus, estimation of senescent cells through P16 or SPIDER staining should be\nperformed in the mice iDTR-ADQ mice to prove the loss of senescent cells as well as in TNFSF11-AdipoCRE mice to prove\nthe persistence of senescent adipo-lineage cells.\nData availability statement is lacking describing the deposition of raw as well as processed data. Making their data set\naccessible, especially a well-annotated Seurat object would have a big impact on the implementation of the data in future\nstudies.\nMinor points:\nThe panels characterizing the histological data, fluorescence as well as H&E or TRAP staining are often way too small.\nQuantification of osteoblasts (1M,S5D, 6I) should be done through measuring osteocalcin positive cells on the bone surface\nand not the total area of staining throughout the marrow space.\nThe in vitro differentiation experiments are misleading, as the isolated cells of Doxo treated mice do not survive or do not\nproliferate to the extent of cells from vehicle treated mice. These data should rather be replaced with proliferation curves and\nCFU-assays but also be part of the supplementary figures.\nIs it correctly described that the gene expression analysis in Supplementary Figure 7J to L has been performed on crushed\nbones depleted for bone marrow? Wouldn’t this imply that the material is devoid of both CAR cells and adipocytes, but still\nthe authors find induction of SASP members? How does this relate to the fact that senescence is restricted to adipo-lineage\ncells?\nThe authors should implement a time line for the diphtheria toxin and tamoxifen treatments, i.e., how many days after\nadipocyte or RANKL ablation die they initiate Doxo or PTX treatment. This would help to understand the timing to establish\nthe basal differences in the iDTR-ADQ and TNFSF11-AdipoCRE mice to their respective controls.\nColor scale in Fig3G is not optimal, since the CAR cells 3 could have a huge white circle for Cdkn2a which is not visible.\nSince the color bar indicates increasing levels, making the color matrix going through white is not optimal.\nThe authors should state the house keeping gene in the figure panels as the method section indicates that different ones\nhave been used.\nThe TNFSF11 induction in the scRNA-seq data should be rephrased, as the majority of CAR cells show down-regulation of\nTNFSF11 while a new population of cells with very high expression levels appear. In addition, a summed score of the Sen-\nMayo gene signature could be used to correlate TNFSF11 expression levels. The latter can demonstrate that CAR cells with\nthis very high TNFSF11 expression levels are also high in senescence-related gene expression.\nThe authors could add a statement on the limitation of the study, i.e., not being able to de-tangle the effect of CAR cells and\nmature adipocytes. In addition, the study aims to prevent bone loss but did not test whether senolytics or interference with\nadipocyte derived RANKL expression after established bone loss can restore bone over time.\nThe observation that TNFSF11-AdipoCRE mice have higher bone mass and loss of osteoclast on trabecular bone surfaces\nraises the question whether adipo-lineage derived RANKL is important for normal bone homeostasis and not only under\npathological conditions, as the authors refer to (line 342). How is site-specificity obtained in a model in which cells distant to\nthe bone surface control osteoclast maturation?\nIf not addressing in the future, the authors should discuss whether baseline adiposity in the marrow cavity affects\nchemotherapy induced bone loss, and if site-specific bone loss corresponds to local adipo-lineage content.\nFinally, there is a trend of inconsistency of how to convey the main message, exemplified by the senolytics and\nsenomorphics treatment. In the first senescence is estimated through the SPIDER assay and co-staining for EBF3 to focus\non CAR cells, while with senomorphics the authors show P16 staining. The first intervention is supported by osteoblast\nnumbers while the latter is through bone formation assays. The effect on RANKL levels are only documented for the\nsenomorphics. This style appeals as existing data have been compiled. For that I would suggest to focus on common\nmeasures in the main figures, but this is clearly a question of taste.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We sincerely appreciate the reviewers' thoughtful evaluation of our manuscript. Their\nconstructive feedback has been invaluable in strengthening the quality and clarity of\nour work. In response, we have carefully addressed each comment in a point-by-point\nmanner that clarifies our findings and further supports our conclusions. However, as\nwe read through the critiques, we feel the main concerns with the work surrounds\nquestions of novelty and the clinical relevance. Given this, we have addressed these\ntwo main critiques immediately below.\nA question of novelty: Our previous work established that senescence was involved\nin chemotherapy-induced bone loss and that a p38MAPK inhibitor could prevent this\neffect. However, at that time, we could not determine the specific cell type undergoing\nsenescence, its location, or the mechanism by which it drove bone loss. The possibility\nremained that the p38MAPK inhibitor’s effect was mediated through limiting\nosteoclastogenesis rather than impacting senescence (SASP) per se. Our current\nmanuscript addresses these fundamental gaps by using complementary genetic and\npharmacologic approaches.\nThe senescence field has been plagued with papers showing that by treating mice\nwith senolytics or activating the suicide gene in the INKATTAC mouse, things got\n“better”. Indeed, until our recent studies showing that senescent fibroblasts impact\ndifferent immune cells based on tumor/tissue context (Ye et al., Can Disc 2024; Belle\net al., Can Disc 2024), almost no paper showed a specific senescent cell target was\ncleared by senolytics or in the INKATTAC mouse. An exception was actually in the\nbone where Khosla’s group showed that osteocytes senesce with age to drive bone\nloss. This is distinct from what we find with chemotherapy where it is CAR cells and\nmature adipocytes that senesce (CAR/BMAds). These results reveal that even within\nthe same tissue (i.e., the bone), the nature of the insult determines the cell type that\nsenesces and the downstream consequences, highlighting, not diminishing the\nbroader biological and clinical significance of our findings. Together, our findings and\nKhosla’s work set a standard for the field by saying that simply showing that\nphenotypes get better upon the elimination of senescent cells without identifying the\ncell type or mechanism responsible is no longer sufficient. Here, through the\ndevelopment of numerous advanced mouse models and extensive experimental\napproaches, we have shown that:\n• in response to chemotherapy, senescence is induced within 9 days and\ncontributes to bone loss.\n• chemotherapy-induced senescence is restricted to CAR/BMAds.\n• senescent bone resident CAR/BMAds are sufficient to drive chemotherapy-\ninduced bone loss.\n• activation of the p38MAPK pathway specifically in CAR/BMAds drives SASP\nand subsequent bone loss.\n• a SASP factor (RankL) produced by senescent CAR/BMAds mediates bone\nloss.\nA question of clinical relevance:\nChemotherapy-induced bone loss is an underappreciated but significant problem, as\ncancer survivors across tumor types face elevated fracture risks and increased long-\nterm mortality. Our work uncovers the mechanism responsible for this adverse effect\nof chemotherapy, providing a direct translational pathway. We show:\n• for the first time that senolytics, which are already in clinical trials for nonlethal\nconditions and have low toxicity profiles, can prevent chemotherapy-induced\nbone loss.\n• importantly that administration of senolytics does not impair the anti-tumor\nefficacy of chemotherapy, addressing a crucial concern for integration into\noncology practice.\n• senescent CAR cells are present in human bone marrow following\nchemotherapy treatment.\nIn summary, our study provides the first detailed cellular and mechanistic insight into\nchemotherapy-induced bone loss and offers a clinically translatable intervention using\nsenolytics—without compromising cancer treatment efficacy. We thank the reviewers\nfor the opportunity to clarify and strengthen the manuscript in these respects.\nBelow, we continue with our point-by-point response to the reviewers’ critiques.\nReviewer #1 indicated that our work provided mechanistic insights into how\nchemotherapy-induced senescence leads to bone loss. Further, Reviewer 1\nhighlighted that we provided compelling results that demonstrated that bone marrow\nadipo-lineage cells drive chemotherapy-induced bone loss. Finally, the reviewer\nindicated that additional strengths included complete and rigorous experiments and\ndata analyses, mechanistic insights, and high clinical relevance. Below we discuss\npoints raised by the reviewer.\nQuestion 1. Can chemotherapy cause direct senescence in osteoclasts (OCs)?\nResponse: This is an interesting question but our work indicates that OC do not\nsenesce. Examination of bone sections from p16-CreERT2/tdTom mice revealed few\ntdTom+ cells on the trabecular bone surface where osteoclasts are located and no\ndifference between vehicle or chemotherapy treated mice was observed (Fig. S6J &\nS6K). In addition, using our ADQ-Cre/Ranklf/f mice, we see clear evidence of p16+\nsenescent cells (Fig. S10P & S10Q) yet OCs are reduced. Because OC do not\nexpress adiponectin (so cannot be killed), these data support our conclusion that the\nreduction in OC is due to loss of the SASP factor, Rankl (Fig. S10M-S10O). To further\nadd to the rigor of our findings, we also created a cell type specific INK model (Fig.\nS8A) that we call the INK-QR mouse. The INK-QR mouse contains a floxed INK allele\nthat allows us to activate the suicide INK gene in a cell type specific manner. We\nmated the INK-QR mouse to the ADQ-Cre mouse and found that we prevent bone\nloss (Fig. 5C & 5D), demonstrating that senescent ADQ+ cells drive bone loss, not\nOC.\nQuestion 2. The statement on “systemic senescent factors do not contribute to bone\nloss” (line 183-184) is overstated.\nResponse: Our data show that bone loss is prevented exclusively in bones\nexpressing the INK allele despite those bones being implanted into wild type mice\nwhose femurs lose bone upon systemic chemotherapy treatment. If systemic factors\nnot originating in the bone drove bone loss, we should not have prevented bone loss\nin the INK vertebrate. Thus systemic factors are not sufficient to drive bone loss.\nHowever, the reviewer does have a point, if the cells in the bone senesce in response\nto a systemic factor and those senescent bone cells drive bone loss, as we show, we\nwould not have detected that in our system. However, given all of our data including\nthe ADQ-Cre/iDTR (Fig. 5A & 5B) and now INK-QR mouse (Fig. 5C & 5D), our data\ndemonstrate that bone loss is driven senescent bone resident cells. However, to avoid\nconfusion, we have removed reference to systemic factors in the updated manuscript.\nQuestion 3: Clarification is needed for what type of sample is used for scRNA-seq.\nResponse: We apologize for not being more precise in the description of the samples.\nAs shown in Fig. 3A, for the scRNA-Seq, we crushed and digested whole femurs\n(containing marrow) and then used MACs sorting to deplete CD45+, Ter119+, CD71+,\nand CD31+ cells and carried out scRNA-Seq on the remaining cells. This approach\nremoved most of the CD45+ immune cells and those that remained were removed in\nthe analyses. This is stated in the legend of Fig. 3A and also clarified this in text.\nQuestion 4: Related to Figure 4\na. In lines 251-252, it states that “no significant changes in p16+/pparγ- cells across\nthe groups”. There are no images in Fig. 4 that support this statement.\nb. In line 271, it states that “there were no changes in Ebf3-SPiDER+ cells in Veh and\nDoxo groups”. What about Ebf3-;tdTom+ cells?\nc. Fig. 4M needs a clear label. Based on the results description, these μCT images\nwere taken from DTRADQ mouse treated with PBS or DT. PBS should be labeled\nabove the first paired μCT images, and DT instead of iDTRADQ should be labeled\nabove the second paired μCT images.\nResponse: In response to question a: we apologize for not initially referencing the\nfigure number; the relevant data are now clearly indicated in Fig. S6E.\nRegarding question b, we have quantified the tdTom⁺ Ebf3⁻ cell population. Our\nanalysis reveals an increased number of tdTom⁺ Ebf3⁻ cells in the Doxo group\ncompared to the Veh group, suggesting that mature adipocytes also express tdTom.\nThe quantification of Ebf3⁻; tdTom⁺ cells have now been included in the revised figure\nFig. S6N.\nIn response to question c, we apologize for the lack of proper labeling. We would like\nto clarify that both DTRf/f and ADQ-Cre+/DTRf/f mice were used in the study, and all\nmice received diphtheria toxin (DT) to assess potential off-target effects. We have now\nclearly indicated this in the images, main text, and figure legends.\nQuestion 5: Related to Figure 5\nIn line 339 and Fig. 5C-F, please clarify which bone samples were used for RT-qPCR,\nbone marrow, crushed bone (depleted bone marrow), or whole bone.\nResponse: Fig. 5C (now 6C) corresponds to an IHC image, not to qPCR. Fig. 5D–\n5F (now 6D-6F) presents qPCR results obtained from bone where marrow was spun\nout (in the revised manuscript, this sample is now referred to as the bone-\nresident fraction) and the bones were then crushed and RNA was isolated. This\napproach is standard in the bone field as it allows for the elimination of the majority of\nCD45+ immune cells. While we acknowledge that some CAR cells/BMAds are\nremoved by this approach, some remain as evidenced by the detection of CXCL12,\nFABP4, and PPARG mRNA (Fig. 1A, shown below). We would like to point out that\nthese experiments were performed before we had discovered that CAR cells/BMAds\nsenesced. For that reason, in Fig. 6J, we returned to our enrichment protocol where\nwe digested whole bone, MACS depleted CD45+ cells and then used FACS sorting to\nisolate Ter119-, CD71-, CD31-, Sca1- and PDGFR+ cells, which significantly\nenriches CAR cells. Finally, Fig. 6G-6I is from BMAds that “floated” to the top of our\ndigested bones, before we subjected the samples to MACs enrichment. Because\nmature BMAds are destroyed upon sorting, this was the most effective way to obtain\nmRNA from them.\nQuestion 6: Related to Figure 6\na. Fig. 6M, IF staining of RANKL. Can\nthe author include a description of the\nlocation of the stained RANKL in text?\nIs it primarily present in the bone\nmarrow space or on the bone?\nb. Line 412-413 states that the\nexpression of other SNAP factors\ndecreased after p38i/MK2i in crushed\nbone depleted bone marrow. Since\nsenCAR/BMAds reside in bone\nmarrow, expression of these SNAPs\nfactors needs to be detected in bone\nmarrow to further confirm the inhibitory\neffect of p38i/MK2i on SNAPs. Fig. 1A. RT-qPCR analysis of gene\nResponse: In response to question expression revealed enrichment of CAR\na, we have now included a description cells and BMAds in the bone-resident\nin the text specifying the location of the fraction, as indicated by elevated Cxcl12,\nstained RankL. FABP4 and PPARG expression.\nIn response to question b, analysis of\nSASP expression was performed on samples where marrow was spun out to reduce\nthe number of CD45+ cells and the residual marrow was crushed (now referred to\nas the bone-resident fraction) and used to isolate RNA. As mentioned above\n(response to point 5), a proportion of CAR cells/BMAds remain as evidence by the\npresence of CXCL12, FABP4 and PPARG mRNA (Fig. 1A, above).\nQuestion 7: Related to Figure 7\na. Representative images are needed for Fig. 7B\nb. For Fig.7E-F. Bioluminescent images and intensity quantification are not sufficient\nto assess tumor burden. Please show histological images of tumor-bearing bones and\nquantify tumor burden by histomorphometry. Histomorphometric analysis is the gold\nstandard for measuring tumor burden in bone.\nc. Fig. 7G. How to distinguish bone loss caused by PyMT-BO-1 cells from paclitaxel\n(PTX)?\nd. In Fig. 7J, tumors are not observed on the histological section, which is inconsistent\nwith the bioluminescent images (showing tumor grew in bones). High-resolution\nimages need to be provided in order to show tumor.\ne. Given that (1) PTX and Doxo may have different anti-tumor effects on breast cancer,\nand (2) senolytic drugs (D+Q) may interact differently with chemotherapies, please\nprovide justification for why PTX, not Doxo, was chosen to use in the breast cancer\nbone metastatic model.\nThe rationale for using the bone metastatic model to support the authors’ findings is\nnot clear. By introducing tumors in the model, it complicates the results and makes it\nhard to evaluate the changes in bone due to tumors or chemotherapy. The author may\nconsider excluding the figure 7 from the manuscript since the model used here is not\nappropriate, and the results do not align with or support the main conclusion. Future\nwork could investigate the author’s findings by using adjuvant breast cancer models.\nResponse: In response to question a, we have added representative images for SA-\nβ-gal staining, now included as Fig. 8B, and have clearly described these findings in\nthe manuscript text.\nFor question b, we have added H & E stains of tumors to complement the BLI\nanalyses BLI (S15A).\nThe reason we choose to use a metastatic model (question c) is that published data\n(Pin et. al., Cancer Lett 2022) showed that primary tumors drive bone loss by\ndisrupting osteocytes. We piloted a primary tumor study by allowing the tumors to\nbecome palpable before the start of chemotherapy to more closely mimic the clinical\nscenario and allow us to measure tumor growth. However, according to this paper, by\nthe time tumors are palpable, the mice would have already experienced bone loss.\nUsing this model, we found that senolytics did not impact the anti-tumor efficacy of\npaclitaxel as expected. Unfortunately, but not surprising given this paper, we found\nonly a trend that bone loss was rescued and we assume that is because tumor-\ninduced bone loss is not senescence dependent. Given these findings we choose to\ntest the metastatic setting and given we rescue the bone loss observed upon\nchemotherapy (Fig. 8I), we can separate the tumor versus chemotherapy-induced\nbone loss. That being said, we have added a sentence to point this caveat out in the\ndiscussion section when we discuss the implications of the data.\nIn response to question d, we have replaced the previous images with new ones that\nmore clearly depict tumor-bearing bones (tumor areas are outlined with black circles,\nFig. 8J).\nFinally, for question e, the rationale for using paclitaxel (PTX) is that PTX is given as\npart of the neoadjuvant (or adjuvant) chemotherapy regimen for all breast cancer\nsubtypes when chemotherapy is indicated. The use of Adriamycin (i.e., doxorubicin)\nis reserved for TNBC, or high-risk ER positive breast cancer and it is rarely used in\nHER2 positive disease. Because our tumor cell lines are luminal B, PTX would be the\nclinically relevant chemotherapy of choice. We have added a statement about this in\nthe text to justify the use of PTX for these studies.\nQuestion 8. Others\na. Detailed methods are needed on how the author defines and measures the OCN+\narea on bone sections. b. In line 271, typo. Fig. 4SL should be Fig. S4L.\nc. The phrase “bone resident cells” is used multiple times throughout the manuscript.\nThe authors seem to use it to refer both to bone marrow cells and cells in/on bone,\nwhich causes confusion. Please clarify it in text or figure legend, as appropriate.\nResponse: In response to question a, we have added detailed information about the\nanalysis of OCN⁺ cells at the end of the immunofluorescence staining methods\nsection.\nFor question b, the typographical error has been corrected.\nIn response to question c, we again apologize for the confusion. We have gone\nthrough the manuscript and now refer to different fractions as follows:\n1. Bone resident enriched fraction contains cells in which MACs kits were\nused to deplete CD45⁻; Ter119⁻; CD71⁻ cells.\n2. CAR cell enriched refers to populations in which we have sorted for CD45-\n, Ter119-, CD31-, Sca1- PDGFR+ cells.\n3. Bone-resident fraction refers to crushed bone where marrow was spun\nout and bone resident and residual CD45+ cells remain. The presence of\nCAR cells/BMAds is ensured by the presence of CXCL12, FABP4 and\nPPARG mRNAs.\nReviewer #2 acknowledged that we used an extensive number of mouse models,\ngenetic and pharmacological approaches to show that senescent bone resident CAR\ncells/BMAds contribute to bone loss through RANK ligand-driven osteoclast\nactivation. In addition, the Reviewer stated that our study “is a well-presented and\nvoluminous article in a hot topic of potential clinical relevance. The experiments are\nsolid and elegantly designed, and the results align with the interest for a broad\naudience.” To strengthen the manuscript the reviewer made several suggestions that\nwe address below.\nQuestion 1: The authors must address the conceptual advance of this work for a\njournal like Nat Comm.\nResponse: This was addressed above.\nQuestion 2: It is well known that osteocyte RANKL is required for cortical bone loss\nwith age and that it can be induced by senescence (Kim et al. JCI insight 2020, PMID:\n32870816).\nResponse: While we are aware of the paper, it should be noted that we do not see\ncortical bone loss in our system nor do we see senescent osteocytes. In addition, in\nthe manuscript from Khosla and colleagues (PMID: 27341653), they found that in\naging, osteocytes senesced and drove bone loss (there was no chemotherapy in this\nstudy). We would argue this supports our argument that identifying the senescent cell\ntype, the stress that induced senescence, and the mechanism(s) by which it impacts\nbiological processes is critical as each can have distinct affects. This is one reason\nour work is novel and provides important conceptual advancement.\nQuestion 3: It seems concerning a recent publication from another laboratory (Ali et\nal. Scientific Reports 2025, PMID: 40389522) reporting that the adverse effects of\nchemotherapy on bone mass are not prevented by senolytics. This point needs to be\nconvincingly addressed. How do the authors explain this apparent discrepancy?\nResponse: We appreciate the reviewer bringing this publication to our attention. The\npaper was published after we submitted our manuscript so at the time of submission,\nwe were unaware of the work. The manuscript reports varied chemotherapy\ntreatments (that differ from our dosing) that were delivered over several weeks to adult\nC57Bl/6 mice, which were of similar age to what we utilized in our studies.\nInterestingly, as we reported in our 2020 Cancer Research paper, after 4 weeks of\ntreatment, we also failed to find senescent markers despite the fact that we could\nprevent bone loss using the INKATTAC mouse. Because we did not find senescence\nmarkers in that first publication, for this study we carried out a time course and dose\ncourse to find the window when senescent cells and the SASP were apparent, which\nwas between 7 and 14 days. It is for that reason we choose the 9-day timeline in our\ncurrent study. Furthermore, we find that our genetic and senolytic approaches prevent\nbone loss but fail to rescue bone that was already lost, which is what was reported in\nthe Ali et. al., paper. We did not include this data in our manuscript because we cannot\nexplain why we cannot rescue bone loss. Thus, we do not think our data is in conflict\nwith the published paper, but instead distinct in that we are examining the effect of\nsenescence on initial bone loss. We have added this to our discussion and referenced\nAli et. al.\nQuestion 4.\nFollowing with the\nprevious point, a\npossibility raised by\nAli and collaborators\nis that the decrease in\nosteoblast numbers is\ndue to chemotherapy\nDDR-induced\nosteoblast apoptosis.\nTherefore,\nmechanistic analyses\nin this manuscript\nmust include a\ncomprehensive\ncharacterization of\napoptosis (e.g.\nTUNEL or activated-\ncaspase 3) damage\nmakers (e.g. gH2AX)\nand how they relate to\nthe crucial cellular\ntypes that maintain\nthe intricate balance\nof bone damage/loss\nFig. 2. A. Immunofluorescence staining for OCN and\nand\nTUNEL demonstrates that osteoblasts do not undergo\nformation/resorption.\napoptosis. B. Quantification of the OCN⁺/TUNEL⁺ area.\nResponse: Per the\nScale bar: 50 μm.\nreviewer’s\nsuggestion, we performed immunofluorescence staining using osteocalcin (OCN) and\nTUNEL. Because DNA damage and subsequent repair and apoptosis can occur over\ntime, we performed TUNEL staining at 1 hour, 2 hours, 6 hours, 24 hours, 72 hours\nand 9 days post-Doxo treatment. Our analysis revealed no significant difference in\nOCN⁺TUNEL⁺ signals between Veh- and Doxo-treated groups in any of the timepoints\nexamined, indicating that apoptosis is unlikely to be the primary mechanism\nunderlying the reduction in osteoblasts following chemotherapy (Fig. 2A and 2B,\nshown above).\nQuestion 5. The authors use the well-established INK-ATTAC mouse model to\nprovide evidence that chemotherapy-induced senescence associates with disrupted\nbone homeostasis, and also p16CreERT2-tdTom mice to identify CARs and BMADs\nwith p16 “senescent” features. To ensure specificity of the p16 antibody the authors\ntested bones from p16KO treated with Doxorubicin. If the authors are correct in their\nhypothesis, I find crucial to validate the main conclusions of this manuscript in\nchemotherapy-treated p16KO mice. What would be the phenotype and mechanism of\nthis model in bone loss and senescence induction?\nResponse: We have extensively used p16 to identify senescent cells and the p16\nminimal promoter to drive our suicide gene (i.e. INK and now INK-QR mouse). We\nhave also used senolytics to complement our studies and kill senescent cells\nregardless of p16 expression. We have no evidence that p16 expression itself is\nimportant for the phenotype we are studying. Furthermore, the relationship between\np16 and SASP is complex. For example, Campisi and colleagues showed that\noverexpression of p16 itself induces a cell cycle arrest reminiscent of senescence but\ndoes not induce the SASP (PMID: 21880712). Further, shRNA directed reductions in\np16 can reduce some SASP components but does not reduce expression to baseline\n(PMID: 33550279). Thus, p16’s role in SASP is not straight forward and it is not clear\nwhat result we would obtain from the p16-KO mouse and how we would interpret it.\nGiven the orthogonal mouse models we have already used including the newly added\nINK-QR mouse, our data indicate that bone resident CAR/BMAds drive bone loss.\nQuestion 6. Also, the authors do not validate the potential role of p21 as senescence\nbiomarker over the manuscript. In fact, the p53/p21 axis is an essential component of\nthe chemotherapy-induced DDR response. Are senCARs and senBMAds positive for\nboth p16 and p21? Or there are different senescent ecosystems? In this regard, district\nsecretomes in p16 and p21 positive senescent cells have been proposed across\ntissues, including the bone (Saul et al. bioRxiv,\nhttps://www.biorxiv.org/content/10.1101/2023.12.05.569858v1.full.pdf).\nResponse: We appreciate the reviewer’s comment and have provided evidence\nshowing that p21 is elevated in bone marrow adipocytes (BMAds), as demonstrated\nin the original Fig. 4E. Additionally, we examined p21 (Cdkn1a) expression in our\nscRNA-seq data, specifically within the CAR cell population (Fig. 3A, below) and CAR\ncell-enriched sample (Fig. 3B, below) and observed a clear upregulation of p21 in\nresponse to Doxorubicin (Doxo). While we recognize that p21 plays an important role\nin senescence within the bone during aging, the key finding from our work remains\nthat elimination of p16⁺ senescent CAR/BMAds is sufficient to prevent all of the bone\nloss, as shown in Figures 1I and 2C. Therefore, while p21 may have unique biology,\nit is unclear what additional insight would be gained by further validating p21 in our\nstudies.\nQuestion 7: Any validation with human datasets / samples post-chemotherapy\nregarding senCARs / senBMAds would reinforce the conclusions and translatability of\nthis study, although I’m aware of the difficulties in getting such samples.\nResponse: As the reviewer points out, it would be difficult if not impossible to obtain\nthese samples from solid tumor patients. Indeed, we have spoken with our clinical\ncolleagues and they feel strongly that the IRB would have ethical concerns and\ntherefore be unlikely to approve an invasive procedure (which this would be) that has\nno direct benefit to the patient. That being said, we were able to identify a dataset from\nbone marrow obtained from leukemia patients fifteen days after chemotherapy\ntreatment and we find that p16 is expressed in CAR cells that lack MKi67 and GSEA\nanalyses reveals a senescence signature. This has been added to Fig. 3I and 3J and\nstrengthens our clinical relevance.\nFig. 3. A. scRNA-Seq dot plot shows elevated p21 expression in Doxo-treated\nCAR cells compared to Veh-treated CAR cells. B. RT-qPCR analysis confirms\nincreased p21 expression in the Doxo-treated group relative to Veh in CAR cell-\nenriched samples.\nOther points-The Discussion is currently underdeveloped and should address all\nconcerns regarding novelty, contradictory data with other groups, and the limitations\nof this study.\n- RNAseq analyses rely on SenMayo datasets, but it would be important to correlate\nthe data with other datasets and senescent signatures.\nResponse: As stated above, at the time of submission we were unaware of the study\nby Ali et. al., and have now added it to our discussion as well as highlighting the\nimportance of identifying the cell type senescing for the field.\nIn regard to the RNA-Seq analyses, it is increasingly recognized that the SASP and\nsenescent transcriptomic signatures vary widely, depending on both the senescing cell\ntype and the senescence-inducing stimulus (PMID: 28844647). Many available\nsignatures were generated by pooling data from diverse cell types or contexts, which\nmay not capture the unique biology of bone-resident CAR/BMAds. Therefore, it is\nimpossible to predict which if any signatures would overlap with ours. Thus, we argue\nthat finding a senescence signature is sufficient and in line with other studies as a\nhypothesis driving exercise, which we confirmed experimentally, the ultimate support\nfor our conclusions that adiponectin positive cells senesce in the bone and drive bone\nloss following chemotherapy.\nReviewer #3\nReviewer 3 felt that our manuscript described “a compendium of in vivo experiments\nusing several genetic mouse lines, that chemotherapy-induced bone loss is linked to\nthe induction of senescence specifically in adipo-lineage cells of the bone of\nchemotherapy induced bone loss”. However, the reviewer also raised some concerns\nthat are addressed below.\nQuestion 1: While depletion of adiponectin expressing cells or TNFSF11(RANKL)\nablation specific to adiponectin expressing cells convincingly shows to prevent\nchemotherapy induced bone loss, the evidence of the senescence signature being\nrestricted to CAR cells and adipocytes is less supported. The latter is mainly due to\nthe usage of a variety of genetic mouse models or staining procedures which are not\ndirectly comparable (often using several methods to stratify the adipo-lineage but\nusing a single approach to deny the role of other bone resident cells). This is surprising\nas the authors performed experiments, such as the adipocyte fractionation and\nscRNA-sequencing in which different compartments could have been compared using\nthe same method to underline the susceptibility of adipocyte-lineage cells to\nsenescence.\nResponse: We apologize as we think that our nomenclature for the cells used for\ndifferent RNA analyses likely caused confusion. As we stated above, we have\nsimplified the nomenclature throughout the manuscript. That being said, we would like\nto emphasize that our data demonstrate that the increase in p16+ expression and SA-\nB-gal is largely restricted to Ebf3+ cells (i.e., CAR cells), which are lost in INK mice\ntreated with AP (Fig. 4K and 4L). However, to conclusively demonstrate that p16+\nadiponectin positive cells are directly responsible for chemotherapy-induced bone loss\nand no other cells that we failed to capture with our orthogonal approaches, we\ncreated the ADQ-Cre/INK-QR mice that allow to activate the senescent suicide gene\nin a cell specific manner (Fig. S8A, schematic). Using these mice, we see loss of\nEbf3+p16+ cells (Fig. S5B and S8C) and restoration of bone upon AP treatment (Fig.\n5C and 5D).\nQuestion 2: Trabecular bone loss has been initially characterized by loss of trabecular\nnumbers; however, this parameter is not changed throughout the intervention studies\nin the INK-ATTAC mice, adipocyte depleted mice, mice lacking RANKL in adipocytes,\nor mice treated with senolytics or senomorphics. In addition, the effects of trabecular\nthickness are limited and therefore are unlikely to explain the observed bone loss.\nDid the authors check mineral content and density to explain the loss of trabecular\nbone without effects on trabecular numbers and thickness.\nResponse: We apologize for not supplying all of these parameters throughout. They\nwere omitted in an effort to streamline the overwhelming amount of data. The BMD\nresults are now presented here for your review (Fig. 4A-4F, below) and also added in\nthe revised manuscript.\nQuestion 3: The authors use a lot of space for the clustering the cell-type identification\nin the scRNA-seq data, but do not follow up on this throughout the manuscript.\nTherefore, clustering could be part of the supplement. In contrast, the gene set\nenrichment analysis should be performed by comparing Doxo condition to vehicle for\neach individual cluster to highlight that CAR cells are more susceptible to the induction\nof senescence than the other cells. For now, the authors compare CAR cells with the\nremaining cells, but not the induction levels in each cluster individually. A heatmap of\nthe Sen-Mayo gene signatures could be used to quantify changes in senescence\nassociated gene transcription.\nResponse: We\nhave moved the\ncell cluster\nmarkers plot to\nthe\nSupplementary\nsection (now\npresented as Fig.\nS5G). As\nsuggested, we\nadded a dot plot\n(Fig. 3F) that\nhighlights\nsenescence\nassociated gene\nsignatures that\nare upregulated\nin Doxo-treated\ncell clusters\nincluding CAR\ncells when\ncompared to Veh-\ntreated cells but\nnot in other cell\ntypes. We believe Fig. 4. A-F. Results show μCT-derived bone mineral density\nthis further (BMD) under indicated treatment conditions.\nemphasizes the\nimpact of Doxo\non CAR cells.\nQuestion 4: Along the line, the fractionation of mature adipocytes for gene expression\n(Fig. 4D) should be complemented by analysis of the SVF fraction, i.e., the cells in the\npellet, to compare induction levels of SASP factors across fractions.\nResponse: As the reviewer suggested, we obtained mRNA from the SVF and\nperformed RT-qPCR and observed no change in p16 expression in the Doxo group,\nwhereas p21 levels were notably elevated (Fig. 5A and 5B, below). The lack of p16\nupregulation in the SVF is likely due to the fact that CAR cells are a minor population\nand was the reason after seeing our scRNA-Seq data, we moved back to an\nenrichment approach. Specifically, we performed qPCR on mRNA from the CAR cell-\nenriched population (CD45- Ter119- CD31- Sca1- PDGFRβ+). In this refined\npopulation, we detected a marked increase in p16 expression in the Doxo-treated\ngroup compared to Veh (Fig. 4C).\nQuestion 5: While the authors emphasize to outline adipo-lineage cells to be highly\nsusceptible to chemotherapy induced senescence and causally involved in bone loss,\nthey do not show how adipocyte ablation and TNFSF11 conditional knockout are\nrelated to the senescence burden within the bone. Thus, estimation of senescent cells\nthrough P16 or SPIDER staining should be performed in the mice iDTR-ADQ mice to\nprove the loss of senescent cells as well as in TNFSF11-AdipoCRE mice to prove\nthe persistence of senescent adipo-lineage cells.\nResponse: As per the reviewer’s suggestion, we have performed\nimmunofluorescence assays to identify senescent cells in bone sections of ADQ-\nCre/iDTR, ADQ-Cre/Rankl, ADQ-Cre/INK-QR, and ADQ-Cre/MK2f/f mice. The results\nare now included in the revised manuscript and shown in Fig. S7F and S7G for the\nADQ-Cre/iDTR model and in Fig. S10M and S10N for the ADQ-Cre/Rankl model. For\nADQ-Cre/INK-QR, and ADQ-Cre/MK2f/f mice it is shown in (Fig. S8B and S8C) and\n(Fig. S14B and S14C) respectively. As shown in these figures, p16+ cells are lost in\nthe ADQ-Cre/iDTR and ADQ-Cre/INK-QR mice while they are retained in the ADQ-\nCre/Rankl mice and ADQ-Cre/MK2f/f mice, as expected.\nQuestion 6: Data availability statement\nis lacking describing the deposition of\nraw as well as processed data. Making\ntheir data set accessible, especially a\nwell-annotated Seurat object would\nhave a big impact on the\nimplementation of the data in future\nstudies.\nResponse: The single cell data was\ndeposited into GEO (GSE289491) at\nthe time of submission and reviewers\nwere granted access to the data. The\nFig. 5. A and B. RT-qPCR analysis\ndata will be released upon publication to\nshows p16 and p21 expression in the\nthe public. In addition, we will upload\nVeh- versus Doxo-treated group in SVF\nour Seurat Objects to GEO so that they\nfraction.\nare also freely available. We apologize\nfor failing to include the statement in the\noriginal submission.\nQuestion 7: The panels characterizing the histological data, fluorescence as well as\nH&E or TRAP staining are often way too small.\nResponse: We can increase the size of the images but will need to increase the figure\nnumbers or move additional data to the supplemental section. We ask for guidance\non which approach is preferred.\nQuestion 8: Quantification of osteoblasts (1M,S5D, 6I) should be done through\nmeasuring osteocalcin positive cells on the bone surface and not the total area of\nstaining throughout the marrow space.\nResponse: We agree with the reviewer and would like to emphasize that for this\nquantification, OCN⁺ signals located within the marrow cavity were deliberately\nexcluded from the analysis. Instead for quantification, OCN⁺ signals proximal to the\nbone surface were included. Importantly, the same analysis criteria and parameters\nwere consistently applied across all treatment and control groups to ensure accuracy\nand comparability.\nQuestion 9: The in vitro differentiation experiments are misleading, as the isolated\ncells of Doxo treated mice do not survive or do not proliferate to the extent of cells\nfrom vehicle treated mice. These data should rather be replaced with proliferation\ncurves and CFU-assays but also be part of the supplementary figures.\nResponse: We thank the reviewer for their comment. The point of the in vitro assays\nwas to show there were fewer cells capable of differentiation given CAR cells are\nprecursors for both osteoblasts and adipocytes. Because we plate the same number\nof living bone marrow cells per well (1 x 104), we believe these data support the\nconclusion that the precursors do not divide. As suggested, these data were moved\nto the supplementary figures.\nQuestion 10: Is it correctly described that the gene expression analysis in\nSupplementary Figure 7J to L has been performed on crushed bones depleted for\nbone marrow? Wouldn’t this imply that the material is devoid of both CAR cells and\nadipocytes, but still the authors find induction of SASP members? How does this relate\nto the fact that senescence is restricted to adipo-lineage cells?\nResponse: As we responded to reviewer 1, we apologize for the confusion created\nby our nomenclature. For these experiments, marrow was spun out but some\nCAR/BMAds remain as evidenced by detection of CXCL12, FABP4 and PPARG\nmRNA (Fig. 1A).\nQuestion 11: The authors should implement a time line for the diphtheria toxin and\ntamoxifen treatments, i.e., how many days after adipocyte or RANKL ablation die, they\ninitiate Doxo or PTX treatment. This would help to understand the timing to establish\nthe basal differences in the iDTR-ADQ and TNFSF11-AdipoCRE mice to their\nrespective controls.\nResponse: As per reviewer suggestion, we have added timelines for ADQ-Cre/DTR\nand ADQ-Cre/Rankl in Fig. S7A and Fig. S10H, respectively. Timelines are usually\nlocated in the supplementary figure due to space but also described in the method\nsection.\nQuestion 12: Color scale in Fig3G is not optimal, since the CAR cells 3 could have a\nhuge white circle for Cdkn2a which is not visible. Since the color bar indicates\nincreasing levels, making the color matrix going through white is not optimal.\nResponse: Based on the suggestion, we have changed the color bar to improve\nvisibility. The new scale goes from red to yellow to blue, without any white, so that\neven low expression levels would be visible. This dot blot shows that Cdkn2a in CAR\ncells 3 is not expressed (see small, blue dot). To emphasize this point, we also\nincreased the dot size scale (from 30 to 60), which made the dot slightly more visible.\nDespite these changes, the figure shows that Cdkn2a is not expressed in this\npopulation. As requested, the image has been added to the revised manuscript (Fig.\n3H).\nQuestion 13: The authors should state the house keeping gene in the figure panels\nas the method section indicates that different ones have been used.\nResponse: This information has been added to the figure legend as per the reviewer’s\ncomments.\nQuestion 14: The TNFSF11 induction in the scRNA-seq data should be rephrased,\nas the majority of CAR cells show down-regulation of TNFSF11 while a new population\nof cells with very high expression levels appear. In addition, a summed score of the\nSen-Mayo gene signature could be used to correlate TNFSF11 expression levels.\nThe latter can demonstrate that CAR cells with this very high TNFSF11 expression levels\nare also high in senescence-related gene expression.\nResponse: We have visualized Tnfsf11 expression in CAR cells 1 using a dot plot,\nwhich clearly illustrates the emergence of a subpopulation with markedly high\nexpression following Doxo treatment (Fig. S10G). Furthermore, we found that\nRANKL⁺ senescent CAR cells exhibit a pronounced senescence-associated\ntranscriptional profile, as demonstrated by the expression of p21 (Cdkn1a) in the dot\nplot and additional senescence-related genes (S10G). These results indicate that the\nhighly RANKL-expressing CAR subpopulation overlaps with cells displaying elevated\nsenescence signatures.\nQuestion 15: The authors could add a statement on the limitation of the study, i.e.,\nnot being able to de-tangle the effect of CAR cells and mature adipocytes. In addition,\nthe study aims to prevent bone loss but did not test whether senolytics or interference\nwith adipocyte derived RANKL expression after established bone loss can restore\nbone over time.\nResponse: We have added a sentence to the discussion that our data does not\nestablish whether it is CAR cells or adipocytes that are dominant (or equivalent) in\nbone loss. Further, as indicated in response to Reviewer 1, we have data that suggests\nonce lost, bone cannot be rescued by eliminating senescent cells.\nQuestion 16: The observation that TNFSF11-AdipoCRE mice have higher bone mass\nand loss of osteoclast on trabecular bone surfaces raises the question whether adipo-\nlineage derived RANKL is important for normal bone homeostasis and not only under\npathological conditions, as the authors refer to (line 342). How is site-specificity\nobtained in a model in which cells distant to the bone surface control osteoclast\nmaturation?\nResponse: We thank the reviewer for the comment. It is true the baseline bone mass\nis higher in the TNFSF11-adipoq-Cre model as was reported by others (PMID:\n34121311), so RANKL is important in normal bone homeostasis as well as\npathological conditions. We have added a statement to the discussion about this as\nrequested. That being said, despite the increased bone mass, loss of RankL in\nadiponectin positive cells make the mice resistant to chemotherapy-induced bone\nloss. To bolster our conclusions, as we stated above, we now have data in the ADQ-\nCre/INK-QR mouse that does not show increased baseline bone mass. Using this\nmodel, we can also prevent chemotherapy-induced bone loss so we feel this\naddresses the concern. This is now presented as (Fig. 5C and 5D).\nQuestion 11: If not addressing in the future, the authors should discuss whether\nbaseline adiposity in the marrow cavity affects chemotherapy induced bone loss,\nand if site-specific bone loss corresponds to local adipo-lineage content.\nResponse: We agree this is an important question but feel it is outside the scope of\nthis study, and an important subject for future investigation.\nQuestion 17: Finally, there is a trend of inconsistency of how to convey the main\nmessage, exemplified by the senolytics and senomorphics treatment. In the first\nsenescence is estimated through the SPIDER assay and co-staining for EBF3 to focus\non CAR cells, while with senomorphics the authors show P16 staining. The first\nintervention is supported by osteoblast numbers while the latter is through bone\nformation assays. The effect on RANKL levels are only documented for the\nsenomorphics. This style appeals as existing data have been compiled. For that I\nwould suggest to focus on common measures in the main figures, but this is clearly a\nquestion of taste.\nResponse: We apologize the inconsistency in staining but would like to clarify the\nreason for the different approaches. When we are looking for loss of p16+ cells (i.e.,\nthe INK-ATTAC mice or ADQ-Cre/DTR or ADQ-Cre/INK-QR mice), we use an antibody\nfor p16, which is from the same species as the Ebf3 antibody. Thus, we cannot use\nthe antibodies for p16 and Ebf3 together. When we wish to show senescent Ebf3\npositive cells are lost, we use co-staining of SPiDER-β-Gal and Ebf3.\nRegarding osteoblast numbers/activity. We first examined changes in osteoblasts by\nmeasuring OCN+ cells in the INK mice and following senolytics. For the\nsenomorphics, which may also translate to the clinic, we wanted to ask how they also\nimpacted osteoblast activity. Finally, we have added RANKL measurements for all\nexperimental approaches where we isolated bone resident enriched cells.\nPlease note, major changes/additions to the text are highlighted in light grey.\nBelow we provide a point-by-point response to each reviewer comment.\nREVIEWER#1\nThe authors have addressed most of my concerns thoroughly. The quality of results\nrelated to the bone metastatic model (Figure 8) has been improved. I thank the author for\nadding the histomorphology assessments to support their findings.\nWe thank the reviewer for this comment.\nAnd, justify the reason for choosing the bone metastatic model over the primary tumor\nmodel. In fact, I think a non-metastatic breast cancer model is more suitable for testing\ntheir hypothesis than a bone metastatic breast cancer model, since it is tricky to\ndistinguish bone destruction caused by chemotherapy or metastatic cancer. Although the\nauthor mentions a paper (Pin et. al., Cancer Lett 2021) states that primary tumors can\nalso cause bone loss through systemic endocrine effects, the results from the paper were\nnot obtained using breast cancer model.\nWe have added a discussion about why we used the metastatic model to test our\ntherapeutic approach and have added the Pin reference to our manuscript. As we stated\nin our discussion, give primary tumor growth can cause bone loss we did not wish to\nconfound our findings with that approach.\nREVIEWER#2\nThe authors have now sufficiently addressed my concerns. I have no further comments\nand congratulate the authors for the work done..\nWe thank the reviewer for their helpful comments.\nREVIEWER#3\nGanes Kumar Raut et al. did a great job in answering the technical as well as conceptual\nconcerns raised during the revision of the manuscript. Importantly, in the revised\nmanuscript the authors made use of their existing data to show that Car cells are\nespecially susceptible to the induction of a senescence gene signature along with\ninduction of Tnfsf11 expression. In addition, they also follow up on the presence of\nsenescent cells when interfering with adipocyte numbers or Tnfsf11 gene expression.\nWe thank the reviewer for their helpful comments.\nMajor:\nThe main finding of the study only provides limited advantage to the field. As outlined in\nthe rebuttal letter, the mechanistic insight is given by the identification of CAR and adipo-\nlineage cells as main cell type being responsible for chemotherapy-induced bone loss\nthrough a senescence-driven induction of RANKL production. While the authors argue\nwith an important statement of: “These results reveal that even within the same tissue\n(i.e., the bone), the nature of the insult determines the cell type that senesces and the\ndownstream consequences, highlighting, not diminishing the broader biological and\nclinical significance of our findings. Together, our findings and Khosla’s work set a\nstandard for the field by saying that simply showing that phenotypes get better upon the\nelimination of senescent cells without identifying the cell type or mechanism responsible\nis no longer sufficient.” One needs to be aware that the induction of senescence\nspecifically in bone marrow adipocytes is linked to glucocorticoid-induced bone loss\n(https://doi.org/10.1016/j.cmet.2023.03.005). The paper by Liu et al. also showed that\ntransfer and elimination of senescent adipocytes can induce and rescue bone loss,\nrespectively, already demonstrating the compartmentalization of senescence within bone\ntissue.\nWe thank the reviewer for pointing out this paper and while it is clear that glucocorticoids\ncan induce bone loss upon transfer, in our setting, we show that it is RANK ligand derived\nfrom endogenous senescent CAR cells that drives bone loss in response to\nchemotherapy.\nDespite its potential clinical relevance, and the clear and thorough execution of\nexperiments, I judge the conceptual novelty to be limited. As such, conceptual novelty\nwould be the identification of molecular cues driving the elevated susceptibility of CAR\ncells and bone marrow adipocytes to chemotherapy induced senescence to understand\ncompartmentalization of senescence within the bone tissue. Or how senescence itself\ntriggers RANKL expression and whether this is distinct from Tnsf11 induction during\nnormal bone remodelling or auto-immune induced bone loss.\nWe thank the reviewer for their opinion and show that it is the activation of the p38MAPK-\nMK2 pathway that leads to expression of Rank ligand in our setting. As to why CAR cells\nare specifically senescing, given the extensive work we have done identifying the\nsenescent cell in the bone and how it drives done loss, we argue that determining why\nCAR cells specifically senescence is beyond the scope of our current manuscript.\nImportantly, I recognize the strength of the data and rigor of the presented study, and I\nfeel comfortable with the editor’s decision to consider this manuscript for publication in\nnature communications.\nWe thank the reviewer for their acknowledgement of our work.\nMinor:\nConceptually, the in vitro differentiation data do neither support the main story line nor\njustify the conclusion taken in the manuscript of an impaired osteoblast differentiation\ncapacity if cell numbers are the primary problem. As outlined in the revision, the authors\nclaim that their data show impairment in proliferation, which is neither the conclusion in\nthe main text nor supported by the assays as increased cell death could have a similar\noutcome. Those data might be removed from the manuscript.\nWe agree that we cannot conclusively conclude if we are seeing reduced proliferation\nand/or apoptosis so simply report the impaired differentiation. Per the editors request, we\nhave left the data in the manuscript.\nThe figures have random styles of colours and shapes, which does not compromise the\nscientific value or message, but might lead to confusion if use of colours is intended.\nNot limited to, but for example Fig 2C versus Fig 2F/2G and S3A-E,M or O for the INK-\nATTAC mice or Fig 5B versus S7M for the ADQ-CRE/DTR mice.\nWe have gone back and attempted to standardize the styles and colors.\nLine 314, it should refer to Figure 5C and 5D instead of 4C and 4D.\nWe have corrected this and thank the reviewer for catching it.\nREVIEWER#4\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe thank the reviewer for their helpful comments.\nREVIEWER#5\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe thank the reviewer for their helpful comments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most of my concerns thoroughly. The quality of results related to the bone metastatic model (Figure 8) has been improved. I thank the author for adding the histomorphology assessments to support their findings.\nOne additional point is that I suggest the author include their pilot experiment on testing the effects of senolytics on\npreventing chemotherapy-induced bone loss using the primary tumor model in supplementary data, as mentioned in their\nresponse to my question 7C. And, justify the reason for choosing the bone metastatic model over the primary tumor model. In\nfact, I think a non-metastatic breast cancer model is more suitable for testing their hypothesis than a bone metastatic breast\ncancer model, since it is tricky to distinguish bone destruction caused by chemotherapy or metastatic cancer. Although the\nauthor mentions a paper (Pin et. al., Cancer Lett 2021) states that primary tumors can also cause bone loss through systemic\nendocrine effects, the results from the paper were not obtained using breast cancer model.", "reviewer_2": "The authors have now sufficiently addressed my concerns. I have no further comments and congratulate the authors for the\nwork done..", "reviewer_3": "Ganes Kumar Raut et al. did a great job in answering the technical as well as conceptual concerns raised during the\nrevision of the manuscript. Importantly, in the revised manuscript the authors made use of their existing data to show that Car\ncells are especially susceptible to the induction of a senescence gene signature along with induction of Tnfsf11 expression.\nIn addition, they also follow up on the presence of senescent cells when interfering with adipocyte numbers or Tnfsf11 gene\nexpression.\nMajor:\nThe main finding of the study only provides limited advantage to the field. As outlined in the rebuttal letter, the mechanistic\ninsight is given by the identification of CAR and adipo-lineage cells as main cell type being responsible for chemotherapy-\ninduced bone loss through a senescence-driven induction of RANKL production. While the authors argue with an important\nstatement of: “These results reveal that even within the same tissue (i.e., the bone), the nature of the insult determines the\ncell type that senesces and the downstream consequences, highlighting, not diminishing the broader biological and clinical\nsignificance of our findings. Together, our findings and Khosla’s work set a standard for the field by saying that simply\nshowing that phenotypes get better upon the elimination of senescent cells without identifying the cell type or mechanism\nresponsible is no longer sufficient.” One needs to be aware that the induction of senescence specifically in bone marrow\nadipocytes is linked to glucocorticoid-induced bone loss (https://doi.org/10.1016/j.cmet.2023.03.005). The paper by Liu et al.\nalso showed that transfer and elimination of senescent adipocytes can induce and rescue bone loss, respectively, already\ndemonstrating the compartmentalization of senescence within bone tissue. Despite its potential clinical relevance, and the\nclear and thorough execution of experiments, I judge the conceptual novelty to be limited. As such, conceptual novelty would\nbe the identification of molecular cues driving the elevated susceptibility of CAR cells and bone marrow adipocytes to\nchemotherapy induced senescence to understand compartmentalization of senescence within the bone tissue. Or how\nsenescence itself triggers RANKL expression and whether this is distinct from Tnsf11 induction during normal bone\nremodelling or auto-immune induced bone loss.\nImportantly, I recognize the strength of the data and rigor of the presented study, and I feel comfortable with the editor’s\ndecision to consider this manuscript for publication in nature communications.\nMinor:\nConceptually, the in vitro differentiation data do neither support the main story line nor justify the conclusion taken in the\nmanuscript of an impaired osteoblast differentiation capacity if cell numbers are the primary problem. As outlined in the\nrevision, the authors claim that their data show impairment in proliferation, which is neither the conclusion in the main text\nnor supported by the assays as increased cell death could have a similar outcome. Those data might be removed from the\nmanuscript.\nThe figures have random styles of colours and shapes, which does not compromise the scientific value or message, but\nmight lead to confusion if use of colours is intended.\nNot limited to, but for example Fig 2C versus Fig 2F/2G and S3A-E,M or O for the INK-ATTAC mice or Fig 5B versus S7M for\nthe ADQ-CRE/DTR mice.\nLine 314, it should refer to Figure 5C and 5D instead of 4C and 4D.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We thank the reviewers for their helpful comments."}]

Decision letter