# Streptomycin targets tumor-initiating cells by disrupting oxidative phosphorylation

Hélène Guillorit, Sébastien Relier, Benjamin Zagiel, Audrey Di Giorgio, Chris Planque, Bastien Felipe, Hélène Hérault, Lucile Bansard, Céline Bouclier, Béatrice Chabi, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Hélène Guillorit, Sébastien Relier, Benjamin Zagiel, Audrey Di Giorgio, Chris Planque, et al. Streptomycin targets tumor-initiating cells by disrupting oxidative phosphorylation. Cell Chemical Biology, 2025, 32 (4), pp.570-585.e7. 10.1016/j.chembiol.2025.03.008. hal-05036262

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Distributed under a Creative Commons Attribution 4.0 International License
# Cell Chemical Biology

## Streptomycin targets tumor-initiating cells by disrupting oxidative phosphorylation

### Graphical abstract

<figure><img src="image_3.png" /></figure>

### Authors

Hélène Guillorit, Sébastien Relier, Benjamin Zagiel, ..., Maria Duca, Françoise Macari, Alexandre David

### Correspondence

francoise.macari@inserm.fr (F.M.),
alexandre.david@inserm.fr (A.D.)

### In brief

In this study, Guillorit et al. identify streptomycin (SM) as an antibiotic that induces mitochondria-dependent cell death in tumor-initiating cells. SM suppresses COX1 expression, disrupting OXPHOS and increasing ROS production. The authors highlight the crucial role of SM's structural features, particularly its aldehyde group, in triggering this cell death mechanism.

### Highlights

* Streptomycin triggers mitochondria-dependent cell death in tumor initiating cells

* Streptomycin inhibits COX1 expression, impairing OXPHOS and increasing ROS production

* Streptomycin structure especially its aldehyde group is crucial for its effects on TIC

* Streptomycin induces a transcriptional program in response to oxidative stress
<figure><img src="image_4.png" /></figure>

## Article

# Streptomycin targets tumor-initiating cells by disrupting oxidative phosphorylation

Hélène Guillorit,<fnref n="1" /> Sébastien Relier,<fnref n="1" /> Benjamin Zagiel,<fnref n="2" /> Audrey Di Giorgio,<fnref n="2" /> Chris Planque,<fnref n="1" />,<fnref n="5" /> Bastien Felipe,<fnref n="1" />

Hélène Hérault,<fnref n="1" />,<fnref n="5" /> Lucile Bansard,<fnref n="1" /> Céline Bouclier,<fnref n="1" /> Béatrice Chabi,<fnref n="6" /> François Casas,<fnref n="6" /> Ornella Clara,<fnref n="1" /> Béatrice Bonafos,<fnref n="6" />

Xavier Mialhe,<fnref n="1" /> Chantal Cazevieille,<fnref n="3" /> Szimonetta Hideg,<fnref n="1" /> Armelle Choquet,<fnref n="1" />,<fnref n="5" /> Amandine Bastide,<fnref n="1" /> Julie Pannequin,<fnref n="1" />

Maria Duca,<fnref n="2" />,<fnref n="7" /> Françoise Macari,<fnref n="1" />,<fnref n="5" />,<fnref n="7" />,\* and Alexandre David<fnref n="1" />,<fnref n="4" />,<fnref n="5" />,<fnref n="7" />,<fnref n="8" />,\*

**SIGNIFICANCE** Despite significant advances in cancer therapy, metastatic disease remains largely incurable, making cancer the leading cause of death worldwide. Increasing evidence suggests that metastasis arises from a subset of disseminated cancer cells with stem-like properties, closely resembling tumor-initiating cells (TICs). Targeting TICs has become a key strategy to prevent cancer recurrence and improve clinical. However, this approach is limited by the inherent resistance of TICs to cell death induced by conventional anticancer therapies. In this study, we identify streptomycin (SM), a bactericidal antibiotic, as a molecule that specifically targets TICs from colon and breast cancer cell lines. SM induces iron-dependent, ROS-mediated cell death, distinct from RSL3-induced ferroptosis, and associated with mitochondrial dysfunction. By inhibiting COX1 expression, SM disrupts the cytochrome c oxidase complex, leading to mitochondrial ROS production. This mechanism is dependent on SM's aldehyde group, as its reduction into dihydrostreptomycin abolishes its effects. These findings reveal a specific action of SM, with the potential to disrupt TIC metabolism and prevent metastatic spread in advanced cancers.

## SUMMARY

Tumor initiating cells (TICs) are the roots of current shortcomings in advanced and metastatic cancer treatment. Endowed with self-renewal and multi-lineage differentiation capacity, TICs can disseminate and seed metastasis in distant organ. Our work identified streptomycin (SM), a potent bactericidal antibiotic, as a molecule capable of specifically targeting non-adherent TIC from colon and breast cancer cell lines. SM induces iron-dependent, reactive oxygen species (ROS)-mediated cell death, which is mechanistically distinct from RSL3-induced ferroptosis. SM-induced cell death is associated with profound alterations in mitochondrial morphology. This effect results from COX1 inhibition, which disrupts the regulation of the cytochrome c oxidase complex and triggers mitochondrial ROS production. SM's aldehyde group is essential, as its reduction into dihydrostreptomycin (DSM) abolishes its activity. These findings reveal a mechanism of action for streptomycin, shedding light on TIC metabolism and resistance, with potential implications for advanced cancer treatment.

## INTRODUCTION

Despite significant advances in cancer therapy, metastatic disease remains incurable in the vast majority of patients and makes

cancer the leading cause of death worldwide.<fnref n="1" /> It is becoming increasingly clear that metastasis originates from a subset of disseminated cancer cells exhibiting stem-like properties and marked similarities with tumor initiating cells (TICs).<fnref n="2" /> TIC

<footnote n="1">¹Institut de Génomique Fonctionnelle, Université Montpellier, CNRS, INSERM, Montpellier, France</footnote>

<footnote n="2">²Université Côte d'Azur, CNRS, Institute of Chemistry of Nice (ICN), Nice, France</footnote>

<footnote n="3">³Institut des Neurosciences de Montpellier (INM), Université de Montpellier, Montpellier, France</footnote>

<footnote n="4">⁴IRMB-PPC, Université de Montpellier, INSERM, CHU Montpellier, CNRS, Montpellier, France</footnote>

<footnote n="5">⁵IRCM, Université de Montpellier, INSERM, Montpellier, France</footnote>

<footnote n="6">⁶DMEM, Université de Montpellier, INRAE, Montpellier, France</footnote>

<footnote n="7">⁷These authors contributed equally</footnote>

<footnote n="8">⁸Lead contact</footnote>

<footnote>*Correspondence: francoise.macari@inserm.fr (F.M.), alexandre.david@inserm.fr (A.D.)</footnote>

<footnote>https://doi.org/10.1016/j.chembiol.2025.03.008</footnote>
phenotype is a plastic condition rather than a defined state, characterized by remarkable adaptation capabilities, self-renewal, and multi-lineage differentiation capacity.³ As such, they can disseminate and re-create a full-fledged tumor in unfavorable distant tissue environment.⁴ For these reasons, targeting TICs became a major goal to eliminate cancer recurrence and improve clinical outcome of patients with advanced cancer. The limitations of this approach reside in the low susceptibility of TICs to cell death mediated by classical anticancer agents. Interestingly, salinomycin, an antimicrobial drug, selectively kills cancer stem-like cells by mediating ferroptosis, a non-apoptotic cell death process driven by reactive oxygen species (ROS) production and iron-dependent lipid peroxidation.⁵ Along the same lines, antibiotics that induce mitochondrial dysfunction of TICs, also represent an appealing strategy for cancer treatment.⁶ Indeed, the mitochondria of TICs display distinctive physical and functional characteristics.⁷ For example, mitochondrial biomass and mitochondria DNA copies are lower in TICs than in other cancer cells, helping to preserve their stem-like properties. Morphologically, TIC mitochondrial cristae are spherical, enlarged, and fragmented, resulting in impaired aerobic function, reduced electron transport chain (ETC) activity and, consequently, lower ROS production. The metabolism of TICs also differs from that of other cancer cells, as they favor oxidative phosphorylation (OXPHOS) over glycolysis to produce the ATP required for their activities.⁷

Cancer cells from either conventional cell lines or patient biopsies can be grown at low cell density in non-adherent conditions and with depleted nutrients. In these unfavorable culture conditions, only a fraction of these cells can survive and form microtumor-like spheroids commonly named tumorspheres. In the case of solid tumor, this cell culture model is often used as a surrogate to evaluate tumorigenic potential.⁸ It is well established that culture of cancer cells in suspension allows the enrichment of TIC whose resilience promotes their survival in unfavorable conditions.⁸ We have previously shown that the penicillin/streptomycin (P/S) cocktail, commonly used in classical cell cultures, impinges on sphere forming ability.⁹ Yet, P/S has no adverse effect on cell proliferation in monolayer cultures, suggesting a selected effect on cell subsets. Accordingly, P/S addition triggers a significant decrease in the number of aldehyde dehydrogenase (ALDH) positive cells. ALDH enzyme activity is widely used as a TIC marker in many solid tumors.¹⁰ Streptomycin (SM), a potent bactericidal antibiotic from aminoglycoside (AG) group, is generally administered for the treatment of individuals with moderate to severe infections. AG belongs to a chemically diverse group of naturally secondary metabolites that bind to specific sites within the ribonucleoprotein complex in bacteria, where they interfere with protein synthesis and ultimately inhibit microbial growth.¹¹ SM structure stands out from other AG. The diaminoinositol unit is a streptidine, instead of 2-deoxystreptamin (2-DOS) or a streptamin, containing two highly basic guanidinium groups. Another molecular feature is the α-hydroxyaldehyde moiety that may create additional interactions with possible targets.

In this study, we identified SM as the sole AG tested capable of targeting non-adherent TIC by inducing mitochondrial-mediated cell death. SM triggers profound mitochondrial dysfunction, ROS production, and intracellular iron pool accumulation without trig-

gering lipid peroxidation, distinguishing its mechanism of action from conventional ferroptosis.¹² Remarkably, SM-resistant cells display a distinctive anti-oxidant gene expression signature that could represent a hallmark of highly adaptable TICs.

## RESULTS

### SM inhibits sphere formation in suspension culture

We have previously shown that the P/S cocktail inhibits sphere-forming ability in suspension cultures.⁹ To identify which of these antibiotics was responsible for this effect, we tested them separately. We used three different human cancer cell lines: SW620 (liver metastasis from colon cancer), CRC1 (derived from patient with primary colon cancer tumor,¹³ and MCF-7 (primary breast cancer). When grown in suspension, these cell lines form tumorspheres in seven to ten days (Figure 1A). By contrast with monolayer cultures, suspension cultures promote the enrichment of TICs, as illustrated by increased mRNA level of five genes involved in stemness (CD133, CD26, CD44, Oct4, and ALDH1A1) (Figure S1A). As expected, SM decreased sphere forming ability in a dose dependent manner in all tested cell lines (Figure 1B), while penicillin alone had no effect (Figure S1B). SM was tested on multiple cancer cell lines, including CTC44, derived in the lab from circulating tumor cells isolated from patient with colorectal cancer,¹³ and three cell lines from different tissue origin (GB4, glioblastoma; A549, lung cancer; and Panc-1, pancreatic cancer). All tested cell lines displayed sensitivity to SM treatment in suspension culture, yet with variable sensitivity (Figures 1C and S1C). GB4 and SW620 were the most sensitive cell lines (IC₅₀ of 100 µM and 120 µM respectively), whereas Panc-1 was more resistant (IC₅₀ around 300 µM). For CRC1 and MCF-7, the analysis revealed an IC₅₀ to 165 µM and 180 µM. In agreement with our previous study, these observations suggest that SM exerts a pan cancer effect on sphere-forming cells, which mostly consists of TICs.¹⁴⁻¹⁶

Next, we sought to understand whether SM impacts on sphere nucleation (i.e., the initiation of sphere formation) or on sphere growth. We treated SW620, CRC1, and MCF-7 cells with SM (200 µM for SW620, 400 µM for CRC1 and MCF-7) at different time points after cell seeding in suspension. Seven days after the beginning of the experiment, spheres were counted (Figure 1D). In all tested cell lines, the maximal effect was obtained when SM was added at the time of cell seeding (day 0). When SM addition was delayed, its effect was gradually lost and SM adjunction became inefficient after day 4. Importantly, we did not notice any change of average sphere size among all tested conditions. These data suggest that SM impacts “sphere nucleation” which relies on TIC phenotype rather than “sphere growth”, fueled by more differentiated cells such as progenitors.

### SM but not DSM inhibits TIC properties both *in vitro* and *in vivo*

Dihydrostreptomycin (DSM) is an antibiotic used in veterinary medicine that shares strong structure similarities with SM and nearly identical bactericidal properties.¹⁷ DSM differs from SM by the reduction of the aldehyde group of the streptose moiety into a primary alcohol group (Figure 2A). Interestingly, whereas this compound keeps a bacterial activity similar to SM, it did not display any effect on sphere-forming ability on SW620,
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 1.** SM inhibits sphere formation in suspension culture</figcaption></figure>

(A) Representative pictures of SW620, CRC1 and MCF-7 cells cultured in monolayer and suspension. Scale bar: 50 µM.

(B) Dose-response effects of SM on sphere forming ability on SW620, CRC1, and MCF-7 in suspension culture. Results are expressed in fold change compared to control and are mean ± SEM of three distinct experiments.

(C) IC₅₀ of SM tested on different cell lines in suspension culture. IC₅₀ were calculated by nonlinear regression, variable slope (four parameters). Results are mean ± SEM of three distinct experiments.

(D) Impact of SM on sphere nucleation. SM at 200 µM (SW620) or 400 µM (CRC1 and MCF-7) was added at different times following cell seeding (day 0, 1, 2, 3, or 4) in suspension culture. Sphere number was quantified at day 7. Results are expressed in fold change compared to control and are mean ± SEM of three distinct experiments.

CRC1, and MCF-7 cell lines at 400 µM (Figure 2B). It is well known that aldehyde-containing drugs can form additional interactions with their target thanks to the formation of a reversible covalent bond with amino groups. This result suggests that the aldehyde group of SM could be involved in the mechanism of action against TICs. We then decided to use DSM as a negative control for all further experiments.

An important feature of TIC properties includes self-renewal ability. We have shown in SW620 cells that SM could interfere with this capacity (Figure 2C). In this experiment, cells were passaged once in suspension culture with either DSM or SM. Upon seven days of culture, spheres (very few for SM condition) were dissociated and the same number of living cells were serially passaged with either DSM or SM. Following seven additional days of culture, the sphere number was evaluated for each condition. SM treatment in the second passage, decreases sphere number by 80% in comparison with DSM-treated control cultures. Interestingly, SM treatment followed by DSM treatment in the second passage did not rescue sphere formation, indicating that SM either alters self-renewal ability of TICs or kills most of them.

Importantly, both SM and DSM did not affect cell growth or viability (Figures S2A and S2B). This substantiates the selectivity of SM over non-adherent TICs in cancer lines from different tissue origins.

Extreme limiting dilution analysis (ELDA) can be used to assess self-renewal capacity of cell populations and has been widely used in the field of cancer stem cells.¹⁸,¹⁹ Briefly, a range of decreasing numbers of tumor cells is cultured and Poisson statistics are used to calculate the frequency of cells capable of initiating spheres. We performed ELDA on SW620 cells treated with either DSM or SM (Figure 2D). As expected, we observed a significant difference in TIC frequency in the presence of SM (a factor of 2,5 for 120 µM and a factor of 3,3 for 200 µM). We performed a complementary *in vivo* experiment based on subcutaneous xenotransplantation of decreasing quantities of cells. We inoculated immunodeficient mice (athymic nude) with suspension cultures of SW620 cells pre-treated or not with an IC₅₀ concentration of SM (Figure 2E). Thirty days later, tumor xenografts (diameter >100 mm³) were counted and harvested. Consistently with ELDA, SM pre-treatment reduced tumor initiation potential by a factor of 2,5 (Figure 2E).

Next, we sought to demonstrate that SM preferentially targets TICs. To do so, we relied on the measurement of ALDH activity, which can be used as a marker to identify and enrich TICs from solid tumors.²⁰ Fluorescence-activated cell sorting (FACS) was used to sort ALDH⁺⁺ and ALDH⁻⁺ cells, which were grown in suspension culture in the presence of increasing quantities of SM or DSM (0–400 µM). After one week, the number of spheres formed for each condition was counted and gave a fairly clear
<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>SW620</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<table><caption>G</caption><thead><tr><th rowspan="2">SW620</th><th colspan="2">TIC frequency</th><th rowspan="2">Fold Change</th><th rowspan="2">P value</th></tr><tr><th>Estimate</th><th>95% Confidence interval</th></tr></thead><tbody><tr><td>DSM 120µM</td><td>1/18</td><td>(1/40-1/9)</td><td></td><td></td></tr><tr><td>SM 120µM</td><td>1/45</td><td>(1/89-1/22)</td><td>2.5</td><td>0.008</td></tr><tr><td>DSM 200µM</td><td>1/27</td><td>(1/64-1/16)</td><td></td><td></td></tr><tr><td>SM 200µM</td><td>1/91</td><td>(1/167-1/51)</td><td>3.3</td><td>0.023</td></tr></tbody></table>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_18.png" /></figure>

<table><caption>Tumor bearing mice</caption><thead><tr><th rowspan="2">SW620</th><th colspan="2">TIC frequency</th><th rowspan="2">Fold Change</th><th rowspan="2">P value</th></tr><tr><th>Estimate</th><th>95% Confidence interval</th></tr></thead><tbody><tr><td>DSM</td><td>1/635</td><td>(1/1545-1/261)</td><td></td><td></td></tr><tr><td>SM</td><td>1/1522</td><td>(1/3676-1/630)</td><td>2.4</td><td>0.199</td></tr></tbody></table>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>SM 1/7</figcaption></figure>

(legend on next page)
result: while ALDH<sup>pos</sup> showed a significant and increasing sensitivity to SM from 150 µM, ALDH<sup>neg</sup> were only sensitive from 400 µM (Figure 2F).

Last, we investigated whether SM injection could inhibit metastasis formation *in vivo*, another hallmark of TICs. Knowing that the liver is the most frequent metastatic site in patients with advanced colorectal cancer,²¹ we used a model of intrasplenic injection to establish colorectal tumors in the mouse liver.²² In brief, we injected SW620 cells into the spleen of severe combined immunodeficiency (SCID) mice, performed splenectomy, treated the animals with SM or DSM (twice weekly), and analyzed tumor formation in the liver after 10 weeks (Figure 2G). It should be noted that mice treated with SM had lower body mass throughout the treatment period (Figure 2G). After 10 weeks, most DSM-treated mice (5 out of 7) exhibited multiple liver metastases, some of them larger than 5 mm. In contrast, only one SM-treated mouse displayed a small tumor in the liver (Figure 2G) suggesting that SM injection is capable of blocking metastasis initiation.

Taken together, these data indicate that SM preferentially targets TICs and their inherent ability to seed tumors in distant organs.

## The inhibition of sphere formation by SM is linked to its distinct chemical structure

SM belongs to the AG class but stands out from other AG members as this compound is the only one having a streptidine genin instead of streptamine or 2-DOS (Figure 3A). Nevertheless, we tested five other AG on their ability to inhibit sphere formation in suspension culture: neomycin, gentamicin, tobramycin, spectinomycin, and paromomycin. These five antibiotics display either 2-DOS genin (neomycin, gentamicin, tobramycin, and paromomycin) or streptamin genin (spectinomycin). In three tested cancer cell lines, these compounds had no effect on sphere formation potential as compared with SM at the same concentrations (Figure 3B). Therefore, the distinctive structure of SM seems to be crucial for its effects on TICs, in particular its aldehyde group as emphasized previously.

In order to better understand the importance of SM structure on sphere formation ability, we have synthesized three SM derivatives (Figure 3C). The first one is streptomycin acid, where the

aldehyde group of the streptose moiety of SM has been oxidized into a carboxylic acid. This compound was prepared starting from commercially available streptomycin upon oxidation in the presence of bromine following a previously reported procedure.²³ Streptomycinic acid was obtained successfully in 73% yield. The two other derivatives represent the two parts of the SM structure: streptidine (the genin containing the guanidine group) and streptobiosamine (the association of the two sugar molecules, streptose and N-methyl-L-glucosamine). These two compounds were obtained upon hydrolysis of streptomycin in acidic conditions ($H_2SO_4$) in 33% and 27% yields, respectively. In SW620 and MCF-7 cell lines, the three synthesized compounds, streptomycinic acid, streptamine and streptobiosamine, did not affect suspension culture at 200 µM (SW620) or 400 µM (MCF-7) as opposed with native SM that abrogates sphere formation within this range of concentration (Figure 3D).

Altogether, these results support the notion that the effect of SM on TICs is not a generic feature of AG but rather a specificity that lies in its peculiar chemical structure.

## SM-resistant cells deploy a transcriptional anti-oxidative stress response program

In order to give us some insights about the molecular mechanisms by which SM prevents sphere formation, we compared gene expression profile from $IC_{50}$ SM-treated cells with DSM-treated cells by means of RNA sequencing (RNA-seq). SW620 and MCF-7 cells were seeded in suspension conditions and total RNA was collected after two days of culture in presence of DSM 120 µM or SM 120 µM and compared to mock-treated cells (CTRL). Bioinformatic analysis did not reveal any change between the control cells and the DSM treated cells. However, we identified an SM-specific gene signature composed of eight genes that were upregulated. (Figures 4A and 4B). Remarkably, all of these genes are associated to cell detoxification processes, suggesting they are involved in cell resistance to SM. The induction of these genes upon SM treatment was confirmed by quantitative polymerase chain reaction (qPCR). Two sets of primers were used for each gene, in order to quantify the level of both mature and immature transcripts. qPCR analysis showed an overexpression of the target genes for both mRNA subtypes (Figure 4C), implying that upregulation results from increased

### Figure 2. SM but not DSM inhibits TIC properties both *in vitro* and *in vivo*

(A) Chemical structure of SM and DSM.

(B) SM or DSM (400 µM) were added in suspension culture and sphere forming ability was quantified. *n* = 3. Mean ± SEM, \*p value < 0.05, \*\*p value < 0.001. Two-sided unpaired t test.

(C) SM impairs self-renewal. SW620 cells were passaged once in suspension culture with either DSM or SM (150 µM). Following 7 days, spheres were dissociated and cells were passaged a second time with DSM or SM. Following seven more days, the sphere number was evaluated. Results are expressed in fold change compared to control (DSM → DSM) and are mean ± SEM of three distinct experiments. \*\*p value < 0.01, \*\*\*p value < 0.001. One-way ANOVA followed by multiple comparisons.

(D) TIC frequency determined by extreme dilution sphere formation assay (ELDA) *in vitro*. SW620 spheres were quantified after 7 to 14 days of culture in the presence of DSM or SM. Results are expressed in fold change compared to control (untreated cells) and are mean ± SEM of five distinct experiments. \*\*p value < 0.01, \*\*\*p value < 0.001. One-way ANOVA followed by multiple comparisons.

(E) Cancer-initiating cell frequencies determined by ELDA assay *in vivo* and estimated within confidence intervals and fold differences between control (DSM) and SM.

(F) SW620 Aldefluor-positive and -negative sphere forming ability after 7 to 14 days of culture in the absence (control) or in the presence of different concentrations of either DSM or SM. Data are expressed as mean ± SEM, *n* = 3 replicates, ns, non-significant, \*p value < 0.05, \*\*p value < 0.01, \*\*\*p value < 0.001. One-way ANOVA followed by multiple comparisons.

(G) Effect of intrasplenic injection of SM on liver metastatic colonization. Mouse body weight (left panel) and number of tumors in the metastatic liver per animal (right panel) are shown. Data are expressed as mean ± SEM for *n* = 7 mice, \*\*p value < 0.01. Lower panel: representative pictures of mouse liver. Scale bars: 0.5 cm.
A

SW620

<figure><img src="image_21.png" /></figure>

B

<table><thead><tr><td>Gene</td><td>Correspondence</td></tr></thead><tbody><tr><td>AKR1C2/ AKR1C3</td><td>aldo-Keto-reductase-2<br>aldo-Keto-reductase-3</td></tr><tr><td>HMOX1</td><td>heme oxygenase-1</td></tr><tr><td>OSGIN1</td><td>oxidative stress induced growth inhibitor-1</td></tr><tr><td>UGDH</td><td>UDP-glucose-6-dehydrogenase</td></tr><tr><td>SQSTM1</td><td>signaling protein p62</td></tr><tr><td>GPX-2</td><td>glutathione peroxidase-2</td></tr><tr><td>TXNRD1</td><td>thioredoxin reductase-1</td></tr></tbody></table>

C

SW620

<figure><img src="image_22.png" /></figure>

MCF-7

<figure><img src="image_23.png" /></figure>

(legend on next page)
transcriptional activity rather from enhanced transcripts stability. Importantly, SM treatment did not upregulate these genes in monolayer culture (Figure S3A). These results suggest that surviving TICs induce a cellular transcriptional program that prevents oxidative stress. The transcription factor NRF2 is well known to regulate cellular antioxidant defense systems and drive cancer progression.²⁴ We therefore investigated whether this factor might be involved in setting this transcriptional program. We assessed NRF2 expression (protein and transcript) (Figures S3B and S3C) and observed no difference following SM treatment. We also silenced NRF2 in SW620 and tested both sphere number and mRNA expression level of four of the upregulated genes (Figures S3D and S3E). No difference was observed following SM treatment.

Transcriptional analysis thus suggests a causal link between cell detoxification ability and resistance to SM. This observation is in line with the ability of AG to target mitochondria and produce ROS.²⁵

## The iron chelator deferoxamine inhibits the effect of SM on sphere formation

To decipher the mechanism by which SM triggers TIC death, we employed specific inhibitors targeting five major cell death types: apoptosis (Z-VAD-FMK), anoikis (Y-27632), necroptosis (necrostatin-1, Nec-1), autophagy (chloroquine, CQ), and ferroptosis (deferoxamine, DFO). Their molecular mechanism of action is described in Figure 5A. These inhibitors were used in three cell lines (MCF-7, SW620, and CRC1) at concentrations that are not toxic for cells in these culture conditions (Figure S4). The iron chelator DFO was the only compound that showed a significant dose-dependent rescue of sphere formation in SM-treated cells in the three tested cell lines (Figures 5B and 5C).

Ferroptosis is a non-apoptotic form of regulated cell death triggered by iron dependent lipid peroxidation.²⁶ In our experiments, Ferrostatin-1 (Fer-1) demonstrated a dose-dependent ability to rescue sphere-forming capacity (Figure 5D). However, Liproxstatin-1 (Lip-1) exhibited toxicity in suspension cultures. To address this, cancer cell lines (SW620, CRC1, and MCF-7) were pretreated in monolayer culture with 1 µM Lip-1 prior to their transfer into suspension cultures supplemented with increasing concentrations of SM. For comparison, cells were also pretreated with 10 µM Fer-1 under the same conditions as a positive control. In all SM-treated conditions, pre-treatment with both inhibitors restored significantly—yet not entirely—sphere formation (Figure 5E). These preliminary observations led us to investigate the possibility that SM could induce TIC death by ferroptosis.

## SM triggers mitochondria-dependent cell death of TIC

Mitochondria, a privileged target of aminoglycosides, play a central role in ROS production as well as in iron metabolism,²⁷ and their implication in ferroptosis has been previously documented.²⁸ We thus evaluated the impact of SM on mitochondrial

ROS production in SW620 and MCF-7 cells seeded in suspension. Staining of both cell lines with MitoSOX Red, a dye commonly used to quantitate mitochondrial superoxide, showed an important increase in mitochondrial ROS production (Figure 6A). Global ROS production was also investigated by CM-H₂DCFDA staining. This dye becomes fluorescent after cellular oxidation and removal of acetate groups by cellular esterases. Our results revealed an increase of H₂DCFDA staining after SM treatment in SW620 (Figure S5A). This suggests that ROS production originates mainly from mitochondria. Consistently, N-acetylcysteine (Nac) and Mito-Tempo (MT), respectively a general antioxidant and a mitochondrial targeted antioxidant, were both able to counteract SM-mediated redox imbalance and restore sphere formation potential (Figures 6B and S5B). Of note, MT was toxic in suspension culture for MCF-7 cells (even at low concentration). Therefore, we modified our protocol accordingly: cells were pretreated with MT (100 µM) in monolayer culture before passaging in suspension cultures with a range of SM concentrations. Tempol (4-hydroxy-TEMPO), a superoxide dismutase mimetic agent that catalyzes the removal of superoxide anions, restored either partially (CRC1 and MCF-7) or totally (SW620) sphere formation ability in SM-treated suspension cultures (Figure S5C). These results indicate that ROS production (especially superoxide) is an important event in SM-induced TIC death mechanism.

Another hallmark of ferroptosis is intracellular iron accumulation. Iron induces the generation of hydroxyl and peroxyl radicals through the Fenton reaction, which promotes lipid ROS production. Calcein-AM and RPA dyes were used to monitor cytoplasmic and mitochondrial free iron pools, respectively. SM treatment of either SW620 or MCF-7 cells resulted in a significant increase in free iron pools in both cellular compartments (Figures 6C and S5D).

Lipid peroxidation is often considered as the final event in the ferroptotic process. We evaluated the levels of lipid peroxidation in SW620 cells by flow cytometry upon incubation with the fluorescent dye BODIPY C11. As a positive control, we employed the small-molecule compound RSL3 that induces ferroptosis via inhibiting the cystine-glutamate antiporter system xc⁻.²⁹ However, neither RSL3 nor SM treatment increased lipid peroxidation in suspension cultures (Figure 6D). This observation aligns with a recent report demonstrating the resistance of suspension cultures to RSL3-induced ferroptosis.³⁰

Finally, we evaluated the role of GPX-4, a major antioxidant enzyme that regulates the cellular redox potential by using GSH to detoxify lipid peroxidation. Dysregulation of GPX-4 in ferroptosis has been largely documented.³¹ However, GPX-4 does not seem implicated in SM-mediated cell death as revealed by our analysis showing no effect on the reduced glutathione (GSH) / oxidized glutathione (GSSG) ratio in SW620 cells (Figure S5E).

Altogether, these results demonstrate that SM triggers mitochondria-dependent cell death of TICs within suspension

### Figure 4. SM-resistant cells deploy a transcriptional anti-oxidative stress response program

(A) DESeq2 analysis of RNA-Seq data, implemented with EdgeR.

(B) Table presenting the 8 upregulated genes in SM treated cells as compared with CTRL or DSM treated cells.

(C) Validation of RNA-seq by qPCR in SW620 and MCF-7 cells. mRNA level of genes was evaluated for mature and immature mRNA. Mean ± SEM of three biological replicates. ns not significant, \*p value < 0.05, \*\*p value < 0.01, \*\*\*p value < 0.001. Two-way ANOVA followed by multiple comparisons.
<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /><figcaption>Figure 5. The iron chelator deferoxamine inhibits the effect of SM on sphere formation</figcaption></figure>

(A) Simplified illustration of the mechanism of action of different cell death inhibitors. Z-VAD-FMK is a pan caspase inhibitor that irreversibly blocks apoptosis. Y-27632 is a selective small molecule inhibitor of Rho-associated kinase (ROCK) involved in cell dissociation-induced anoikis. Necrostatin-1 (Nec-1) is a

(legend continued on next page)
cultures. This mechanism, while sharing common features with ferroptosis such as iron accumulation and ROS production, appears to be distinct from it.

## SM disrupts COX1 expression and induces mitochondrial dysfunction

Recent report suggests that antibiotics targeting prokaryotic ribosome could specifically impact mitochondrial biogenesis and activity in TIC and trigger cell death.32,34 In order to test the impact of SM on mitochondrial content, we first employed MitoTracker Green (MTG) staining followed by flow cytometry analysis. The cells were seeded in suspension cultures and treated with either DSM or SM at their respective IC50 concentration (120 µM for SW620 and 180 µM for MCF-7). After six days (SW620) or ten days (MCF-7) of culture, MTG staining revealed no significant difference between SM- and DSM-treated cells (Figure 7A). In addition, quantitative PCR analysis of mitochondrial DNA (mtDNA) copy number confirmed that total mitochondrial mass was not altered by SM (Figure S6A).

Next, we monitored the effect of SM on mitochondrial membrane potential (MMP). We employed tetramethylrhodamine methyl ester (TMRE) staining, a cell permeant, positively charged dye that accumulates in active mitochondria. In SW620 and MCF-7 suspension culture, TMRE staining followed by flow cytometry showed a significant increase in MMP polarization in SM-treated cells as compared to DSM treated cells (Figure 7B). We assessed mitochondrial morphology using transmission electron microscopy (TEM) in SW620 cells cultured in suspension with DSM or SM (120 µM) for six days. In SM treated cells, TEM revealed severe alterations in mitochondrial morphology with mitochondrial swelling and cristae vanishment (Figure 7C). By contrast, SM treatment of monolayer culture at higher concentration (400 µM) did not promote any morphological difference (Figure S6B). The connection between mitochondria swelling and SM-induced cell death was also strengthened by TEM observations showing significant restoration of mitochondrial morphology when SW620 cells were cultured in suspension in presence of MT (Figure 7C).

The mechanisms leading to mitochondrial swelling are still poorly understood, although ions, pHi, and MMP are likely the main players regulating matrix swelling.35 We hypothesized that the osmotic imbalance between the cytosol and the matrix was due to a malfunctioning of the mitochondrial OXPHOS complexes. OXPHOS subunits are encoded on either the nuclear or

the mitochondrial genome. SM has been shown to bind the mitochondrial ribosome, potentially impairing the synthesis of mitochondria-encoded respiratory chain proteins.36,37 We assessed the expression of several proteins present in OXPHOS complexes from SM- or DSM-treated spheres (Figure 7D): NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9), NADH-ubiquinone oxidoreductase chain 2 (ND2), NADH-ubiquinone oxidoreductase chain 2 (ND4), succinate dehydrogenase [ubiquinone] flavoprotein subunit (SDHA), and cytochrome c oxidase subunit 1 (COX1). While the levels of nuclear-encoded proteins (NDUFA9 and SDHA) remained largely unchanged, COX1 expression, encoded by the mitochondrial genome, was significantly reduced. Notably, this effect was specific to COX1, as two other mitochondrially encoded proteins (ND2 and ND4) were not affected by SM treatment. Additionally, the mRNA levels for COX1 remained unchanged (Figure S6C), indicating that COX1 expression is inhibited at the post-transcriptional level, possibly through specific inhibition of translation.

COX1 is a subunit of the cytochrome c oxidase complex (complex IV), the last enzymatic complex in the mitochondrial electron transport chain, whose role is to catalyze oxidative phosphorylation.38 As expected, the reduced expression of COX1 in the presence of SM coincided with a marked reduction in COX enzymatic activity (Figure 7E).39

Finally, we employed the oxygraph-2k respirometer (Oroboros) to estimate mitochondrial respiratory capacity in DSM- or SM-treated spheres (Figures 7F, 7G, and S6D). SM-treated cells showed increased respiration (as a proportion of maximum capacity [ETS] in basal mode [routine]) as well as in the presence of oligomycin (leak), suggesting the presence of increased proton leakage at the inner membrane.

These findings indicate that the impact of SM on mitochondria is selective, targeting COX1 without affecting ND2 or ND4. The inhibition of COX1 is likely sufficient to initiate a cascade reaction, resulting in the production of ROS.

## DISCUSSION

In this study, we show that SM targets specifically non-adherent TICs and promotes their death. We unraveled a transcriptional program that likely contributes to cell detoxification and SM resistance. We demonstrate the importance of SM structural features in triggering this mechanism of cell death, in particular

RIP1-targeted inhibitor of necroptosis. Chloroquine. (CQ) prevents autophagosome formation during autophagy process. The iron chelator deferoxamine (DFO) is a ferroptosis inhibitor by preventing iron accumulation.

(B) DFO inhibits the effect of SM on sphere formation. Sphere number after 7 days of co-treatment of cells with SM and increasing concentrations of different cell death inhibitors. Results are expressed in fold change compared to control. n = 3 biological replicates. Mean ± SEM, **p value < 0.01, ***p-value < 0.001. One-way ANOVA followed by multiple comparisons.

(C) DFO rescues sphere forming ability in dose-dependent manner. Sphere number after 7 days of co-treatment of cells with SM and increasing concentrations of DFO. Results are expressed in fold change compared to control. n = 3 biological replicates. Mean ± SEM, **p value < 0.01, ***p value < 0.001. One-way ANOVA followed by multiple comparisons.

(D) Ferrostatin-1 (Fer-1) rescues sphere forming ability in dose-dependent manner. Sphere number after 7 days of co-treatment of cells with SM and increasing concentrations of Fer-1. Results are expressed in fold change compared to control. n = 3 biological replicates. Mean ± SEM, *p value < 0.1, **p value < 0.01. One-way ANOVA followed by multiple comparisons.

(E) Ferroptosis inhibitor Liproxstatin-1 (Lip-1) counteract SM effect on sphere formation. After 72 h of pretreatment in adherent condition with Lip-1 (1 µM) or Fer-1 (10 µM), cells were passaged in suspension culture with increasing concentrations of SM. Results are expressed in fold change compared to control. n = 3 biological replicates. Mean ± SEM, *p value < 0.05, **p value < 0.01, ***p value < 0.001. Two-way ANOVA followed by multiple comparisons.
<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /><figcaption>**Figure 6.** SM triggers mitochondria-dependent cell death of TICs</figcaption></figure>

(A) Flow cytometry quantification of MitoSOX Red mean fluorescence intensity in cells cultured in suspension condition. Results are expressed in fold change compared to control. n = 3 biological replicates. Mean ± SEM, \*p value < 0.05. Two-sided unpaired t test. Histogram representative of triplicates.

(B) ROS inhibitors restore sphere formation ability. Left panel: sphere number after 7 days of co-treatment of SW620 cells with SM and increasing concentrations of either N-acetylcysteine (Nac) or Mito-Tempo (MT). Results are expressed in fold change compared to control. n = 3 biological replicates. Mean ± SEM, \*\*p value < 0.01, \*\*\*p value < 0.001. One-way ANOVA followed by multiple comparisons. Right: Sphere number after MCF-7 pretreatment with MT for 3 days, then passaged in suspension culture with increasing concentrations of SM. Mean ± SEM, \*p value < 0.05. Two-way ANOVA followed by multiple comparisons.

(C) Mitochondrial iron pool is increased after SM treatment in suspension culture. Flow cytometry quantification of RPA mean fluorescence intensity in cells cultured in suspension with IC₅₀ concentrations of either DSM or SM. Results are expressed in fold change compared to control. n = 3 biological replicates. Mean ± SEM, \*p value < 0.05, \*\*p value < 0.001. Two-sided unpaired t test. Histogram is representative of three independent experiments.

(D) Flow cytometry quantification of Bodipy-C11 positive cells in SW620 cultured in monolayer or suspension condition with RSL3 for 2H, DSM and SM for 3 days (monolayer culture) or 7 days (suspension culture). Results are expressed in fold change compared to control. n = 3 biological replicates. ns not significant. Mean ± SEM, one-way ANOVA followed by multiple comparisons. The histogram illustrates one representative biological replicate.
Mitotracker Green

<figure><img src="image_36.png" /><figcaption>A</figcaption></figure>

SW620

<figure><img src="image_37.png" /></figure>

MCF-7

<figure><img src="image_38.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_39.png" /></figure>

SW620

<figure><img src="image_40.png" /></figure>

TMRE

<figure><img src="image_41.png" /></figure>

MCF-7

<figure><img src="image_42.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_44.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_45.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_46.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_47.png" /></figure>

(legend on next page)
the aldehyde group. Chemical reduction of the former in DSM provided us an ideal control for *in vitro* and *in vivo* experiments. Finally, we characterized SM-induced cell death and elucidated its mode of action, which is clearly dependent on ROS production and iron accumulation, two key hallmarks shared with ferroptosis. Mechanistically, our findings reveal that SM specifically inhibits COX1 expression, leading to impaired OXPHOS activity and subsequent ROS production.

While sharing certain characteristics with ferroptosis, several factors indicate a distinct cell death mechanism. The most striking difference is the absence of lipid peroxidation, reinforced by the idea that sphere forming cells are resistant to classical RSL3-induced ferroptosis, as emphasized by a recent publication in lung adenocarcinoma cancer stem cells.³⁰ Further, GPX4, a key regulator of ferroptosis,³¹ does not appear to be involved in SM-induced cell death, as the GSH/GSSG ratio remains unchanged and GPX4 is not among the genes associated with resistance to SM treatment. It is important to note, however, that GPX4-independent ferroptosis mechanisms have been identified.⁴⁰ Nevertheless, SM-induced cell death may represent a distinct form of cell death, exhibiting certain hallmarks of ferroptosis, including its dependence on iron accumulation and ROS.

Our results raise an important question regarding the mechanism by which SM triggers mitochondrial dysfunction. A recent report solved the structure of the complex of streptomycin with the mitochondrial ribosome using high-resolution single-particle cryoelectron microscopic (cryo-EM) structures.⁴¹ This enabled them to highlight the fact that the aldehyde group ends up in the hydrated gem-diol state once in the cell, and participates in potential interactions with rRNA. DSM, with its reduced aldehyde group, could not reproduce this type of interaction, which could have a different impact on mitochondrial ribosome function. It is therefore conceivable that SM could subtly—and selectively—alter mitochondrial translation activity, which involves only 13 proteins. This selectivity would lead to a reduction in mitochondrial translation of COX1 and subsequent dysfunctional respiratory chain activity, without having any significant impact on mitochondrial content (as we have observed). Indeed, mitochondrial content relies essentially on the expression of proteins encoded in the nucleus and translated in the cytoplasm. This hypothesis is supported by the observation that COX1 inhibition is accompanied by increased expression (although not significant, Figure 7D) of

ND2 and ND4, suggesting a redistribution of ribosomes onto other mitochondrial transcripts. Nevertheless, how can we explain the specificity of SM activity toward TIC? One potential explanation lies in the specificity of TIC metabolism and their cellular environment,⁴² which could affect aldehyde chemistry, thus altering SM association with mitochondrial rRNA. A better understanding of this mechanism will require a comparative study (TICs vs. non-TICs) of the impact of the cell medium on streptomycin chemistry.

Taken together, our data lead us to propose the following sequence of events. Firstly, SM inhibits COX1 expression and induces dysfunction of complex IV. The result is increased production of superoxide anions due to a reaction between untransferred electrons and oxygen. The highly unstable superoxide anion then reacts with iron (Fenton reaction) to produce the hydroxyl radical, which ultimately causes severe mitochondrial oxidative stress. Cells resistant to SM display an adaptive response to oxidative stress, resulting in a detoxification program that enables them to limit ROS production and prevent cell death. Complex IV dysfunction also prompts them to increase respiration by proton leakage and therefore cope with the increased ROS production via an uncoupling phenomenon, as observed in our respirometer data.

In the course of this study, we uncovered a transcriptional program that likely counteracts oxidative stress and allows the survival of a subpopulation of SM-treated TICs. Remarkably, this inducible program was NRF2-independent and restricted to eight genes involved in cell detoxification and/or ferroptosis inhibition. For instance, AKR1C2 and AKR1C3 are aldo-keto-reductases involved in NADPH-dependent reductions. HMOX1 has been associated with systemic iron homeostasis, stress response,⁴³ and more recently with ferroptosis.⁴⁴ Most certainly, enhancing expression of this specific set of genes requires a very selective transcriptional activator such as, for example, ZNF410, the NuRD component CHD4.⁴⁵ Our findings pave the way for future studies aiming at identifying this factor for the design of therapeutic strategies targeting oxidative stress resistant cells such as TICs.

Most importantly, our study reveals a mechanism of action of SM that could be exploited in cancer biology. Since SM is already an FDA-approved drug used in clinic, it represents an ideal candidate for drug repositioning in anti-TIC therapy. Furthermore, SM has a pan-cancer activity, which reinforces its value in clinic. While side effects, such as ototoxicity, can

Figure 7. SM disrupts COX1 expression and induces mitochondrial dysfunction

(A) Flow cytometry quantification of Mitotracker Green mean fluorescence intensity in cells cultured in suspension with either DSM or SM. Results are expressed in fold change compared to control. n = 3 biological replicates. Mean ± SEM, ns not significant. Two-sided unpaired t test. The histogram is a representative biological replicate.

(B) Flow cytometry quantification of TMRE mean fluorescence intensity in SW620 and MCF-7 cells treated with DSM or SM in suspension culture. CCCP has been used as a positive control in each experiment. Results are expressed in fold change compared to control. n = 3 biological replicates. Mean ± SEM, \*p value < 0.05, \*\*p value < 0.01, \*p value < 0.001. One-way ANOVA followed by multiple comparisons. The histogram illustrates one representative biological replicate.

(C) Representative ultrastructure of mitochondria from SW620 treated with DSM or SM at 120 µM with or without addition of MT in suspension culture by TEM. Scale bar: 500 nm.

(D) Western blot analysis showing expression levels of mitochondrial complex I (NDUFA9, ND2, ND4), complex II (SDHA) and complex IV (COX1). n = 3 biological replicates. Mean ± SEM, \*p-value < 0.05. Two-sided unpaired t test.

(E) Effect of SM on cytochrome c oxidase (COX) activity. n = 6 biological replicates. Mean ± SEM, \*p value < 0.05. One-way ANOVA followed by multiple comparisons.

(F) Effect of SM or DSM on respiration of SW620 cells. Mitochondrial respiration is expressed as oxygen flux per million cells [pmol·s⁻¹·¹⁰⁻⁶ cells].

(G) Mitochondrial respiration is expressed as flux control ratios of routine and leak over ET capacity. Mean ± SEM of 5 independent cultures.
be put forward to limit its use, preventing tumor spreading in advanced (e.g., stage 3 colorectal cancer) cancer patient remains a matter of life and death. Further, combined with chemotherapy, SM could also be of use to prevent bacterial infections in neutropenic patients.

## Limitations of the study

Repositioning SM for advanced cancer treatment is appealing due to its ability to target TICs. However, the concentrations required to achieve this effect in vivo may trigger side effects such as ototoxicity. Therefore, developing streptomycin-derived compounds with greater efficacy against TICs while exhibiting reduced toxicity would be beneficial. In this study, we modified the aldehyde group through reduction and oxidation to assess its importance for biological activity. Streptomycin is a complex aminoglycoside that, to the best of our knowledge, has not been chemically modified before. Due to its structural complexity, common protection and deprotection strategies are challenging to apply.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Alexandre David (alexandre.david@inserm.fr).

### Materials availability

All unique/stable and non-commercially available reagents generated used in this study can be requested from the lead contact in reasonable amounts with a completed materials transfer agreement.

### Data and code availability

* RNA-seq data have been deposited at Gene Expression Omnibus (GEO) and are publicly available as of the date of publication. Accession number is listed in the key resources table.

* All data reported in this paper will be shared by the lead contact upon request.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

This work was supported by INCa (PLBio 2017-160), Ligue Contre le Cancer (AAP ARN-2022.LCC/AID), SIRIC Montpellier Cancer (INCa-DGOS-Inserm 6045), and Canceropole Grand Sud-Ouest (Grant Emergence). We thank Montpellier Genomix sequencing facility (http://www.mgx.cnrs.fr), particularly Hugues Parinello, Anaïs Louis, and Xavier Mialhe, as well as the iExplore animal facility, especially Amelyne David and Oceane Gentilini. We also acknowledge MRI (Montpellier Rio Imaging), particularly Cécile Dejou, and ARPEGE. We thank the MitO2 platform (INRAe - UMR DMEM) that belongs to Montpellier Alliance for Metabolomics and Metabolism Analysis (MAMMA), Biocampus Montpellier. Special thanks to Jean-Emmanuel Sarry (CRCT, Toulouse) and Laurent Le Cam (IRCM, Montpellier) for their helpful insights.

## AUTHOR CONTRIBUTIONS

A.D., M.D., F.M., and H.G. designed experiments and analyzed the results; H.G., S.R., B.Z., A.D.G., C.P., H.H., B.F., L.B., C.B., B.C., F.C., O.C., B.B., C.C., S.H., A.C., and A.B. performed experiments; A.D., M.D., F.M., H.G., X.M., and J.P. performed data analysis; A.D., M.D., F.M., and H.G. wrote the manuscript; all the authors reviewed the final version of the manuscript.

## DECLARATION OF INTERESTS

The authors declare no conflict of interest.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS**

* Cell lines and culture condition

* Animals

* **METHOD DETAILS**

* Sphere formation assay

* Proliferation assay

* Cytotoxicity assay

* *In vitro* extreme limiting dilution assay

* *In vivo* experiment

* Flow cytometry

* RNA extraction and quantitate PCR (RT-qPCR)

* Mitochondrial DNA copy number assessment

* Mitochondrial labeling with MitoTracker green FM

* Measurement of mitochondrial membrane potential

* Transmission electron microscopy

* ROS production assessment

* Mitochondrial respiration

* Cytochrome c oxidase (COX) activity

* C11 BODIPY 581/591 staining

* Cytoplasmic and mitochondrial iron pool

* GSH/GSSG ratio detection

* RNA-sequencing

* Sequencing data analysis

* Cell extracts and western blotting analysis

* Chemistry: Synthesis of streptomycin analogs

* Synthesis of streptomycin acid

* Synthesis of streptidine and streptobiosamine

* **QUANTIFICATION AND STATISTICAL ANALYSIS**

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.chembiol.2025.03.008.

Received: November 28, 2023

Revised: February 12, 2025

Accepted: March 19, 2025

Published: April 9, 2025

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<figure><img src="image_49.png" /></figure>

## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Anti-MTCO1 antibody [1D6E1A8]</td><td>Abcam</td><td>Cat# ab14705; RRID:AB_2084810</td></tr><tr><td>Anti-NDUFA9 antibody [20C11B11B11]</td><td>Abcam</td><td>Cat# ab14713, RRID:AB_301431</td></tr><tr><td>Anti-SDHA antibody [2E3GC12FB2AE2]</td><td>Abcam</td><td>Cat# ab14715, RRID:AB_301433</td></tr><tr><td>Anti-Nrf2 antibody</td><td>Abcam</td><td>Cat# ab137550, RRID:AB_2687540</td></tr><tr><td>Anti-ND2 antibody</td><td>Proteintec</td><td>Cat# 19704-1-AP, RRID:AB_10638920</td></tr><tr><td>Anti-ND4 (A-16)-R</td><td>Santa Cruz Biotechnology</td><td>Cat# sc-20499-R, RRID:AB_2149745</td></tr><tr><td>Anti-Actin antibody (clone AC-40)</td><td>Sigma-Aldrich</td><td>Cat# A4700, RRID:AB_476730</td></tr><tr><td>m-IgGκ BP-HRP</td><td>Santa Cruz Biotechnology</td><td>Cat# sc-516102, RRID:AB_2687626</td></tr><tr><td>mouse anti-rabbit IgG-HRP</td><td>Santa Cruz Biotechnology</td><td>Cat# sc-2357, RRID:AB_628497</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>N2 Supplement</td><td>Gibco</td><td>Cat#17502-048</td></tr><tr><td>hBasic-FGF</td><td>Miltenyi Biotec</td><td>Cat#130-093-564</td></tr><tr><td>DMEM</td><td>Miltenyi Biotec</td><td>Cat#130-093-564</td></tr><tr><td>DMEM-F12 (1:1) GlutaMAX</td><td>Gibco</td><td>Cat#31331-028</td></tr><tr><td>hEGF</td><td>Miltenyi Biotec</td><td>Cat#130-097-751</td></tr><tr><td>Insulin</td><td>Sigma</td><td>Cat#I9278</td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>Cat#G7513</td></tr><tr><td>Accumax</td><td>Sigma</td><td>Cat#A7089</td></tr><tr><td>B-27 supplement</td><td>Gibco</td><td>Cat#12587010</td></tr><tr><td>BSA</td><td>Sigma</td><td>Cat#A7906</td></tr><tr><td>Poly-HEMA</td><td>Sigma</td><td>Cat#P3932</td></tr><tr><td>Streptomycin</td><td>Sigma</td><td>Cat#S9137</td></tr><tr><td>Dihydrostreptomycin</td><td>Sigma</td><td>Cat#D5155</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>Cat#G1264</td></tr><tr><td>Tobramycin</td><td>Sigma</td><td>Cat#T1783</td></tr><tr><td>Neomycin</td><td>Sigma</td><td>Cat#N1876</td></tr><tr><td>Spectinomycin</td><td>Sigma</td><td>Cat#S4014</td></tr><tr><td>Paromomycin</td><td>Sigma</td><td>Cat#P9297</td></tr><tr><td>N-acetylcystein</td><td>Sigma</td><td>Cat#A9165</td></tr><tr><td>Mito-TEMPO</td><td>Sigma</td><td>Cat#SML0737</td></tr><tr><td>Tempol</td><td>Sigma</td><td>Cat#176141</td></tr><tr><td>Z-VAD-FMK</td><td>Promega</td><td>Cat#G7231</td></tr><tr><td>Necrostatin-1</td><td>Sigma</td><td>Cat#N9037</td></tr><tr><td>Y-27632</td><td>Sigma</td><td>Cat#Y0503</td></tr><tr><td>Chloroquine diphosphate salt</td><td>Sigma</td><td>Cat#C6628</td></tr><tr><td>Deferoxamine</td><td>Sigma</td><td>Cat#D9533</td></tr><tr><td>Liproxstatin-1</td><td>Sigma</td><td>Cat#SML1414</td></tr><tr><td>Ferrostatin-1</td><td>Sigma</td><td>Cat#SML0583</td></tr><tr><td>Sulforhodamine B</td><td>Sigma</td><td>Cat#S1402</td></tr><tr><td>Tris-Base</td><td>Fisher Bioreagents</td><td>Cat#BP1521</td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>Cat#1.00063</td></tr><tr><td>Tris-Reagent</td><td>Sigma</td><td>Cat#T9424</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>Cat#I9516</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>M-MMLV</td><td>Invitrogen</td><td>Cat#28025</td></tr><tr><td>Random hexamers</td><td>Invitrogen</td><td>Cat#N8080127</td></tr><tr><td>SYBR Green master mix</td><td>Roche</td><td>Cat#04 707 516 001</td></tr><tr><td>dNTP</td><td>Invitrogen</td><td>Cat#R0192</td></tr><tr><td>Mitotracker Green FM</td><td>Invitrogen</td><td>Cat#M751</td></tr><tr><td>TMRE</td><td>Sigma</td><td>Cat#87917</td></tr><tr><td>CM-H2DCFDA</td><td>Invitrogen</td><td>Cat#C6827</td></tr><tr><td>MitoSOX Red</td><td>Invitrogen</td><td>Cat#M36008</td></tr><tr><td>BODIPY™ 581/591 C11</td><td>Invitrogen</td><td>Cat#D3861</td></tr><tr><td>RPA</td><td>Squarix biotechnology</td><td>Cat#ME043</td></tr><tr><td>FCS</td><td>Eurobio</td><td>Cat#CVFSVF00</td></tr><tr><td>TCA</td><td>Sigma</td><td>Cat#T6399</td></tr></tbody></table>

Critical commercial assays

<table><tr><td>Bradford protein assay kit</td><td>Bio-Rad</td><td>Cat#5000001</td></tr><tr><td>GSH/GSSG-Glo Assay kit</td><td>promega</td><td>Cat#V6611</td></tr><tr><td>Universal Plus mRNA-seq kit</td><td>Tecan Trading AG, Switzerland</td><td>N/A</td></tr><tr><td>KAPA Library quantification kit</td><td>Roche, Basel, Switzerland</td><td>N/A</td></tr></table>

Deposited data

<table><tr><td>RNA-seq data</td><td>This paper</td><td>GEO: GSE292379</td></tr></table>

Experimental models: Cell lines

<table><tr><td>CRC1</td><td>biopsies provided by CHU-Carémeau (Nîmes, France)</td><td>ClinicalTrial.gov Identifier #NCT01577511</td></tr><tr><td>CTC44</td><td>Isolated from metastatic chemotherapy-naïve stage IV colorectal patients. Grillet F. et al.<sup>13</sup></td><td>N/A</td></tr><tr><td>SW620</td><td>ATCC</td><td>Cat#CCL-227</td></tr><tr><td>HT29</td><td>ATCC</td><td>Cat#HTB-38</td></tr><tr><td>GB4</td><td>isolated from a human GB (male)</td><td>N/A</td></tr><tr><td>MCF7</td><td>ATCC</td><td>Cat#HTB-22</td></tr><tr><td>Panc-1</td><td>ATCC</td><td>Cat#CRCL-1469</td></tr></table>

Experimental models: Organisms/strains

<table><tr><td>CB17/Icr-Prkdcscid/IcrIcoCrl</td><td>Charles river</td><td>White(albino) 236</td></tr></table>

Oligonucleotides

<table><tr><td>Primers used for qPCR: see Table S1</td><td>This paper</td><td>N/A</td></tr></table>

Software and algorithms

<table><tr><td>ImageJ</td><td>NIH LOCI</td><td>v2.14.0/1.54f<br>https://imagej.nih.gov/ij/</td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>V10.4.0<br>https://www.graphpad.com/</td></tr><tr><td>FlowJo</td><td>BD Bioscience</td><td>V10.10.0<br>https://www.flowjo.com/</td></tr><tr><td>Flowing software</td><td>Flowing software</td><td>(v 2.5.1; http://flowingsoftware.btk.fi/)</td></tr><tr><td>LightCycler480</td><td>Roche</td><td>1.4.9.0803</td></tr></table>

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Cell lines and culture condition

Patient-derived cell culture of colon cancer cells CRC1 were obtained from primary tumor of colorectal biopsies provided by CHU-Carémeau (Nîmes, France, ClinicalTrial.gov Identifier #NCT01577511) within an approval protocol (ethical agreement no
<figure><img src="image_50.png" /></figure>

2011-a01141-40). Signed informed consents were obtained from patients prior to samples acquisition in accordance with all ethical and legal aspects. CTC44 are circulating tumor cells derived from blood of metastatic chemotherapy-naïve stage IV colorectal patients.¹³ SW620 (ATCC, CCL-227™ donor sex: male), HT29 (ATCC, HTB-38™ donor sex: female), MCF-7 (ATCC, HTB-22™ donor sex: female), Panc-1 (ATCC, CRL-1469 donor sex: male) and A549 (ATCC, CRM-CCL-185 donor sex: male) cells were cultured at 37°C under humidified 5% CO2, in DMEM (Gibco) media supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Eurobio) and 2 mM glutamine. For suspension culture, cells were plated at 100 cell/100µL in M11 medium (DMEMF12 (1:1) GlutaMAX medium, N2, Glucose 0.3%, insulin 20 µg/mL, hBasic-FGF 10 ng/mL, hEGF 20 ng/mL) in low attachment plates treated with Poly-HEMA at 10 mg/mL. GB4 cells were isolated from a human (donor sex: male, WHO grade IV) GB resected at Gui de Chauliac Hospital in Montpellier and were described by Guichet et al.⁴⁶ GB4 were cultured as described,⁴⁷ on poly-HEMA coated vessels and in defined media (DMEM/F12 1:1 GlutaMAX medium (Invitrogen), D-Glucose 0.3% (sigma) N2 Supplement (Invitrogen), B-27 supplement (Gibco), 10 ng/mL EGF (Peprotech) and 10 ng/mL FGF2 (Peprotech).

## Animals

Six-week-old female CB17/Icr-Prkdcscid/IcrIcoCrl nude mice were obtained from Charles River Laboratories (Wilmington, MA, USA). All experiments were performed according to the European Union (Council directive 86/609EEC) and institutional/local guidelines on laboratory animal usage. Animal protocols were approved by the French ethical committee for animal testing (authorization referral #1627). Mice were randomly assigned to experimental groups.

## METHOD DETAILS

### Sphere formation assay

Cells were plated at a density of 100 cells per 100 µL in M11 medium (DMEM/F12 (1:1) GlutaMAX, N2 Supplement, 0.3% glucose, 20 µg/mL insulin, 10 ng/mL hBasic-FGF, and 20 ng/mL hEGF) in low-attachment 96-well plates pre-treated with Poly-HEMA at 10 mg/mL. After 7 days, spheres with a diameter greater than 50 µm were counted.

### Proliferation assay

Cell proliferation was monitored in real-time using the xCELLigence® system E-Plate. Two thousand cells were seeded in wells with DSM or SM at 400 µM in DMEM medium. The impedance value of each well was monitored by the xCELLigence® system for duration of 72 h and expressed as a cell index value.

### Cytotoxicity assay

A thousand of cell were seed in 96-well plate with decreasing doses of DSM or SM (1/3 dilution from 2 mM). After 96 h, cells were fixed for at least 1 h at 4°C in 10% TCA. After two washes with Milli-Q water, cells were incubated in 0.4% Sulforhodamine B (SRB) solution for 20 min at room temperature followed by three washes with acetic acid 1% solution. Absorbance was measured at 560 nm after resuspension in 10 mM Tris-Base.

### In vitro extreme limiting dilution assay

SW620 cells were grown in suspension with or without 120 µM SM for 7 days. The frequency of cancer cells with *in vitro* stem cell potential was determined by Extreme Limiting Dilution Analysis (ELDA)⁴⁸ after dilution in M11 medium of 1000/100/10 and 1 cell per well in 96-well plates in the absence (control) or in the presence of 120 µM or 200 µM of either DSM or SM. The number of wells containing at least one sphere (>50µm) was counted after 7 to 14 days. Cancer cell frequency was determined using online ELDA (extreme limiting dilution analysis) software (https://bioinf.wehi.edu.au/elda/software).

### In vivo experiment

All experiments were performed according to the European Union (Council directive 86/609EEC) and institutional/local guidelines on laboratory animal usage. Animal protocols were approved by the French ethical committee for animal testing (authorization referral #1627).

### Extreme limiting dilution assay (ELDA)

SW620 cells were grown in suspension with or without 120 µM SM for 7 days. After spheres dissociation, living FACS sorted cells were suspended in 100µl icecold Matrigel (ozyme)/ DMEM (v : v). Cells (100 – 500 - 2 500) were subcutaneously injected into CB17 SCID nude mice (6 weeks, females, 5 mice per group). Tumor volume ([length × width²]/2) were monitored twice a week up to 12 weeks using a caliper and groups killed when tumor reached 1 500 mm³. Tumor apparition frequency was determined using online ELDA (extreme limiting dilution analysis) software (https://bioinf.wehi.edu.au/elda/software).

### Intrasplenic injection for liver metastasis induction

SW620 cells were grown in suspension with 120 µM SM or DSM for 3 days 100 000 cells were injected in the spleen of CB17 SCID nude mice (6 weeks, females, 7 mice per group). After induction of anesthesia with isofluran (VETFLURANE®, Virbac, France) and subcutaneous buprenorphine (0.1 mg/kg; Reckitt Benckiser, Slough, UK) for postoperative pain relief, mice were placed on heating pad and the upper abdomen was shaved and sterilized with 70% ethanol solution. A 1 cm flank incision was made to expose the tip of the spleen, which was gently withdrawn from the abdomen. Using a 27-gauge needle, 50µL of cell solution containing 100 000 cells
were gently injected underneath the splenic capsule over 180 s in order to rule out fluid extravasation and spleen rupture. After needle withdrawal, splenectomy was then performed by ligation with silk sutures on splenic vessels. Peritoneum and muscles were sutured closed with one stitch and the wound was closed with a clip.⁴⁹ Mice were treated twice a week with SM or DSM at 150mg/Kg in PBS and sacrificed at 10 weeks after tumor injection. Health status of mice was monitored once a week by body weight and clinical examination. Animals were sacrificed by cervical dislocation under general anesthesia and size and number of liver metastasis were evaluated.

### Flow cytometry

The ALDH activity of adherent cells was measured using the ALDEFLUOR kit (Stem Cell Technologies), according to the manufacturer's instructions. ALDH-positive and ALDH-negative cells were identified by comparing the same sample with and without the ALDH inhibitor diethylaminobenzaldehyde (DEAB).⁵⁰ Stainings were analyzed using the MACSQUANT (Miltenyi) analyzer and the Flowing software (v 2.5.1; http://flowingsoftware.btk.fi/). To exclude nonviable cells, Sytox blue was added at a concentration of 0.1 µg/ml.

### RNA extraction and quantitate PCR (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer instructions. For RT-qPCR analyses, 1 µg of RNA was reverse-transcribed into cDNA using random hexamers (Invitrogen) and 1 U of MML-V reverse transcriptase (Invitrogen). Quantitative gene expression was performed using SYBR Green master mix (Roche) on LightCycler 480 Instrument (Roche). Results were normalized to GAPDH or actin expression and analyzed using the ΔΔCt method. Primer sequences are listed in the key resources table and in the supplementary data.

### Mitochondrial DNA copy number assessment

Quantification of the mtDNA copy number relative to the nuclear DNA (ncDNA) was realized using real-time PCR as previously described.⁵¹ Real-time quantitative PCR to quantify mtDNA and ncDNA was performed on 10 ng nucleic acids, directly on total RNA isolated by TRIzol, by using the SYBR Green master mix (Roche) and the LightCycler 480 instrument (Roche), with the following program: 10 min at 95°C, 50 cycles of 10 s at 95°C, 15 s at 67°C and 15 s at 72°C. ncDNA was quantified by using the following primers: β2-microglobulin-F: 5'-TGCTGTCTCCATGTTTGATGTATCT -3'; β2-microglobulin-R: 5'-TCTCTGCTCCCCACCTCTAAGT -3'. Total mtDNA was quantified by amplifying a DNA domain within the D-loop of mtDNA by using the following primers: Universal-F: 5'-TTAACTCCACCATTAGCACC-3'; Universal-R: 5'-GAGGATGGTGGTCAAGGGA-3.

### Mitochondrial labeling with MitoTracker green FM

After treatment with DSM and SM (120 µM for SW620, 180 µM for MCF-7), cells were cultured in suspension condition for 6 days (SW620) or 10 days (MCF-7) and spheres were dissociated with Accumax. Fifty thousand cells were stained with 50nM MitoTracker Green FM for 10 min at 37°C and the fluorescence emission was detected by flow cytometry.

### Measurement of mitochondrial membrane potential

Level of MMP was measured with tetramethylrhodamine methyl ester (TMRE) probe. Cells were treated in suspension with SM or DSM (120 µM for SW620, 180 µM for MCF-7). After 6 days (SW620) or 10 days (MCF-7), spheres were dissociated and fifty thousand cells were stained with 20 nM TMRE at 37°C for 15 min. Excess TMRE was removed by washing the cells once with PBS/BSA 1% and analysis was done following flow cytometry.

### Transmission electron microscopy

Tissues were immersed in a solution of 2.5% glutaraldehyde in PHEM buffer (1X, pH 7.4) overnight at 4°C. They were then rinsed in PHEM buffer and post-fixed in a 0.5% osmic acid + 0.8% potassium Hexacyanoferrate trihydrate for 2 h at dark and room temperature. After two rinses in PHEM buffer, the cells were dehydrated in a graded series of ethanol solutions (30-100%). The tissues were embedded in EmBed 812 using an Automated Microwave Tissue Processor for Electronic Microscopy, Leica EM AMW. Thin sections (70 nm; Leica-Reichert Ultracut E) were collected at different levels of each block. These sections were counterstained with uranyl acetate 1.5% in 70% Ethanol and lead citrate and observed using a Tecnai F20 transmission electron microscope at 120 kV in the Institut des Neurosciences de Montpellier: Electronic Microscopy facilities, INSERM U1298, Université Montpellier, Montpellier France.

### ROS production assessment

Cells were seeded in suspension culture with SM or DSM at 120 µM (SW620) or 180 µM (MCF-7) for 6 days (SW620) or 10 days (MCF-7). Total ROS generation was measured with CM-H₂DCFDA (Invitrogen) and mitochondrial ROS were assessed with MitoSOX Red (Invitrogen). Spheres were dissociated with Accumax and fifty thousand cells were incubated with 50 nM (SW620) or 500 nM (MCF-7) CM-H₂DCFDA or 5 µM MitoSOX at 37°C for 10 min. Cells were harvested and analysis was done following flow cytometry.
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### Mitochondrial respiration

SW620 cells were seeded in suspension culture for 7 days with DSM or SM at 120 µM. Spheres were dissociated with Accumax, and collected in 1ml of MIR05 respiration medium (0.5mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 3mM MgCl2, 60mM Lactobionic acid, 20mM taurine, 10mM KH2PO4, 20mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 110m Mucrose, and 1 g/L bovine serum albumin, pH 7.1). Cells were counted with a hemocytometer after staining with trypan blue to evaluate dead cells. A total of 2 × 10⁶ cells were transferred in two sealed thermostated chambers (37°C) of high-resolution oxygraph (Oxygraph-2k, OROBOROS Instruments) containing 2ml of MIR05 respiration medium. Basal respiration (Routine, R) was measured, then proton leak-associated respiration (Leak, L) was determined with titration of oligomycin (2µl; 5 µM titration). Maximal respiration i.e. maximal Electron Transfert (ET) capacity (ETS) was determined with titration of carbonylcyanide- 3-chlorophenylhydrazone (CCCP), a chemical uncoupler, into the cell suspension (1 µl; 0.5 µM titration). In the end, antimycin a (5 µM) was added to determine residual nonmitochondrial oxygen consumption (ROX). Data acquisition and analysis were performed using Oxygraph-2k DatLab software version 7.4.0.4 (OROBOROS Instruments).

### Cytochrome c oxidase (COX) activity

SW620 cells were cultured in suspension culture for 7 days with DSM or SM at 120 µM. Spheres were dissociated and pellet was incubated 10 min in Phosphate buffer (K2HPO4, 8 mM, pH 7.4) and then lysed by three cycles of freezing/thawing in liquid nitrogen. Protein amount was determined using a Bradford protein assay kit. The activity of cytochrome oxidase is measured with a spectrophotometer following the oxidation of reduced cytochrome c at 550 nm, pH 7.4, and 37°C. Cytochrome c was reduced with sodium dithionite.⁵²

### C11 BODIPY 581/591 staining

SW620 cells were grown in monolayer or suspension culture with or without 120 µM SM. After 3 days (monolayer culture) and 7 days (suspension culture), untreated cells were incubated for 2 h with RSL3 prior to lipid peroxidation analysis. Spheres were dissociated and 100,000 cells were incubated with 2 µM C11-BODIPY (581/591) at 37°C for 15 min. Subsequently, the cells were harvested, resuspended, and analyzed by flow cytometry.

### Cytoplasmic and mitochondrial iron pool

The cytoplasmic chelatable iron pool was assessed using Calcein-AM and the mitochondrial iron pool was detected using Rhodamine B-[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester (RPA), both specific Fe²⁺ specific fluorescent sensors. After treatment of cells with DSM or SM (120 µM for SW620, 180 µM for MCF-7) during 6 days (SW620) or 10 days (MCF-7) following sphere dissociation with Accumax, fifty thousand cells were harvested and incubated with 10 nM Calcein-AM or 1 µM RPA for 15 min at 37°C. Following two washes with PBS/BSA 1%, cytoplasmic and mitochondrial iron pool were measured by flow cytometry.

### GSH/GSSG ratio detection

SW620 cells were seeded in suspension culture for 6 days with DSM or SM at 120 µM. After sphere dissociation with Accumax, five thousand cells were plated in white 96-well plate. GSH and GSSG were quantified using the GSH/GSSG-Glo Assay kit (Promega), according to the manufacturer's protocol. Luminescence in relative light unit (RLU) was measured with a plate reader (Tecan Infinite M200) and the following formula was used to calculate GSH/GSSG ratio: (Net treated total glutathione RLU - Net treated GSSG RLU) / (Net treated GSSG RLU / 2).

### RNA-sequencing

SW620 cells were cultured in suspension for 2 days in 3 biological replicates (Control, DSM 120 µM, SM 120 µM). Total RNA was extracted using TRIzol Reagent (Invitrogen) following manufacturer's procedure. Quality and quantity were evaluated by electrophoretic migration and spectrometry. 200 ng total RNA per sample was sequenced by the platform MGX.

Library was prepared using the Universal Plus mRNA-seq kit (Tecan Trading AG, Switzerland) according to the manufacturer's instructions. Briefly, polyadenylated RNAs were selected using oligo-dT magnetic beads. PolyA+ RNAs were fragmented using divalent cations at elevated temperature and reverse transcribed using random hexamers, reverse transcriptase and actinomycin D. Deoxy-TTP was replaced by dUTP during the second strand synthesis to prevent its amplification by PCR. The double stranded cDNAs were repaired and adenylated at their 3' ends then ligated to Tecan adaptors including UDs. Ligated cDNAs were submitted to a strand selection prior to PCR amplification for 15 cycles. PCR products were purified using AMPure XP Beads (Beckman Coulter Genomics, Brea, CA, USA). The size distribution of the resulting libraries was monitored using a Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA) and the libraries were quantified using the KAPA Library quantification kit (Roche, Basel, Switzerland). Libraries were denatured with NaOH, neutralized with Tris-HCl and diluted to 300 pM. Clustering and sequencing were performed on a NovaSeq 6000 (Illumina, San Diego, CA, USA) using the 100nt single read protocol on an S2 flow cell to generate 50 to 84 million sequences.

### Sequencing data analysis

Image analyses and base calling were performed in real time by the Real-Time Analysis software (Illumina). Demultiplexing was performed using Illumina's conversion software (bcl2fastq 2.20). The quality of the raw data was assessed using FastQC (v0.11.9) from
the Babraham Institute and the Illumina software SAV (Sequencing Analysis Viewer). FastqScreen (v0.15.1) was used to identify potential contamination.

Before alignment, adapter sequences were trimmed from the reads using Cutadapt (v3.4). A splice junction mapper, TopHat2 (v2.1.1)⁵³ (using Bowtie v2.3.5.1),⁵⁴ was used to align the RNA-Seq reads to the human genome (Hg38) with a set of gene model annotations (gtf file downloaded from NCBI on 2020-08-30). Final read alignments having more than 6 mismatches were discarded. Samtools (v1.9) was used to sort and index the alignment files. Then, gene counting was performed with featureCounts v2.0.0.⁵⁵ As the data is from a strand-specific assay, the reads have to be mapped to the forward strand of the gene (-s 1 option). Before statistical analysis, genes with less than 15 reads (cumulating all the analyzed samples) were filtered out.

Differentially expressed genes were identified using the Bioconductor⁵⁶ package DESeq2 v1.26.0 (R version 3.6.2).⁵⁷ Data were normalized using the DESeq2 normalization method. Genes with adjusted p-value below 5% (according to the FDR method from Benjamini-Hochberg) were considered differentially expressed.

Biological validation of RNA-Seq results was performed using quantitative Polymerase Chain Reaction (qPCR) (primer sequences in Table S1).

## Cell extracts and western blotting analysis

Cells were lysed in ice-cold RIPA buffer (Tris 50 mM, sodium chloride 15 mM, sodium desoxycholate 0,25 %, NP40 1 %, sodium orthovanadate 1 mM, sodium fluoride 10 mM, β-glycerophosphate 10 mM, EGTA 2 mM, DTT 1 mM, cOmplete EDTA-free protease inhibitor cocktail 5 X). Samples were separated on 12 % SDS-polyacrylamide gels, blotted on nitrocellulose membranes, blocked using PBS-Tween 0,1 % with either 5 % (w/v) semi-skimmed milk or 5 % (w/v) BSA for one hour at room temperature and probed either overnight at 4°C or for one hour at room temperature with antibodies described in the key resources table. Revelation was performed using ECL Prime or Select reagents (Cytiva) with a Chemidoc MP imager (Bio-Rad).

## Chemistry: Synthesis of streptomycin analogs

Reagents and solvents were purchased from Merck and Carlo Erba Reagents and used without further purification. Streptomycin and dihydrostreptomycin were purchased from Merck as sulfate salts. HPLC was performed using a Water Alliance 2695 pump coupled with Waters 996 photodiode array detector and a Phenomenex Gemini (5 µm, NX-C18, 4.6 × 250 mm Column for analytical HPLC and 10 x 150 mm for semipreparative HPLC). All HPLC analyses were run at room temperature using a gradient of CH3CN containing 0.1% TFA (eluent B) and water containing 0.1% TFA (eluent A) from 5% to 100% of B in 30 min at a flow rate of 1 mL/min for the analytical column and 3.5 mL/min for the semipreparative column. 1H and 13C NMR spectra were recorded on a Bruker AC 400 MHz spectrometer. Chemical shifts are reported in parts per million (ppm, δ) referenced to the residual 1H resonance of the solvent (D2O, δ 4.79). Splitting patterns are designated as follows: s (singlet), d (doublet), m (multiplet). Coupling constants (J values) are listed in hertz (Hz). Low resolution mass spectra (MS) were obtained with a Brucker Daltonics Esquire 3000+ electronspray spectrometer equipped with API ionization source. High resolution mass spectra (HRMS) were obtained with a LTQ Orbitrap hybrid mass spectrometer with an electronspray ionization probe (Thermoscientific, San Jose, CA) by direct infusion from a pump syringe to confirm correct molar mass and high purity of compounds.

## Synthesis of streptomycinic acid

Streptomycinic acid was synthesized starting from commercially available streptomycin sulfate (100 mg, 0.137 mmol) in one step upon treatment with Br₂ (0.02 mL, 0.388 mmol, 2.8 eq.) in H₂O (1.5 mL). The reaction mixture was stirred 5 days at room temperature and in the dark, then quenched using sodium thiosulfate. After filtration, the obtained filtrate was concentrated under reduced pressure and purified by semipreparative HPLC leading to desired streptomycinic acid in 73% yield (59.6 mg). Retention time 5.3 min); ¹H NMR (400 MHz, D₂O) δ = 5.30–5.20 (m, 2H), 4.60–4.50 (m, 2H), 3.90–3.80 (m, 2H), 3.75–3.65 (m, 2H), 3.50–3.30 (m, 7H), 3.30–3.20 (m, 1H), 2.72 (s, 3H), 1.12 (d, J = 5.0 Hz, 3H); ¹³C NMR (125 MHz, D₂O) d = 173.6, 158.1, 99.4, 93.5, 86.3, 81.4, 78.8, 74.7, 72.5, 71.5, 70.8, 69.3, 68.8, 60.5, 59.6, 58.9, 30.8, 12.2; HRMS (ESI): *m/z* 598.26807 (M+H)+ (C₂₁H₄₁O₁₃N₇ requires 598.26786).

## Synthesis of streptidine and streptobiosamine

Commercially available streptomycin was hydrolyzed to streptidine and streptobiosamine following a previously reported procedure (J. Biol. Chem. 1966, 241, 3142). Streptomycin (100 mg, 0.137 mmol) was dissolved in 1N sulfuric acid (1 mL) and stirred 48 h at 37°C. The reaction mixture was then left 24h at 4°C to obtain a colorless precipitate of streptidine sulfate in 33% yield (20.7 mg). Retention time 7.7 min; ¹H NMR (400 MHz, D₂O) δ = 3.50–3.35 (m, 4H), 2.90–2.80 (m, 1H), 1.30–1.20 (m, 1H); MS (ESI): *m/z* 263.14624 (M+H)+ (C₈H₂₀O₄N₆ requires 263.14623). Acetone (1 mL) was then added to the filtrate and the solution left 24h at 4°C to precipitate the remaining streptidine. Lyophilization of the filtrate led to the obtention of streptobiosamine as a colorless solid in 27% yield (12.5 mg). Retention time 6.5 min; ¹H NMR (400 MHz, D₂O) δ = 5.46 (d, J = 2.0 Hz, 1H), 5.00–4.95 (m, 1H), 4.40–4.30 (m, 2H), 3.90–3.75 (m, 4H),
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3.60-3.40 (m, 2H), 3.30-3.20 (m, 1H), 2.81 (s, 3H), 1.16 (d, J = 5.0 Hz, 3H); MS (ESI): m/z 370.17050 (M+MeOH)+ (C14H28NO10 370.17132).

<figure><img src="image_53.png" /></figure>

QUANTIFICATION AND STATISTICAL ANALYSIS

All graphs and statistical analyses were performed using GraphPad Prism Software (v8.4.2) and shown in the figure legends. Two-
sided unpaired T-test was used when comparing two groups. One-way analysis of variance (ANOVA) was used when comparing
three or more groups. Two-way ANOVA was used for multiple comparisons within groups. Graphs display mean ± standard
deviation. P-values are denoted as non-significant (ns), \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. Statistical test methods were reported
in figure legends.
Supplemental information

Streptomycin targets tumor-initiating cells
by disrupting oxidative phosphorylation

Hélène Guillorit, Sébastien Relier, Benjamin Zagiel, Audrey Di Giorgio, Chris Planque, Bastien Felipe, Hélène Hérault, Lucile Bansard, Céline Bouclier, Béatrice Chabi, François Casas, Ornella Clara, Béatrice Bonafos, Xavier Mialhe, Chantal Cazevieille, Szimonetta Hideg, Armelle Choquet, Amandine Bastide, Julie Pannequin, Maria Duca, Françoise Macari, and Alexandre David
A

SW620

<figure><img src="image_54.png" /></figure>

CRC1

<figure><img src="image_55.png" /></figure>

MCF-7

<figure><img src="image_56.png" /></figure>

B

SW620

<figure><img src="image_57.png" /></figure>

CRC1

<figure><img src="image_58.png" /></figure>

MCF-7

<figure><img src="image_59.png" /></figure>

C

HT29

<figure><img src="image_60.png" /></figure>

CTC44

<figure><img src="image_61.png" /></figure>

GB4

<figure><img src="image_62.png" /></figure>

A549

<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /><figcaption>**Figure S1: SM inhibits sphere formation in suspension culture, related to Figure 1.**</figcaption></figure>

A.) TIC markers are enriched in suspension culture. mRNA level of genes involved in stemness was evaluated by RT-qPCR in SW620, CRC1 and MCF-7 cells cultures in monolayer or suspension conditions in absence of antibiotic. Mean ± SEM of three biological replicates. ns not significant, ***p-value < 0.001. Two-way Anova followed by multiple comparisons. B.) Penicillin G does not impact sphere forming ability. Increasing concentrations of Penicillin G were added in suspension cultures of SW620, CRC1 and MCF-7. Spheres were counted following 7 days. Results are expressed in fold change compared to control and are mean ± SEM of three distinct experiments. ns not significant, ***p-value < 0.001. One-way Anova followed by multiple comparisons. C.) Dose-response effects of SM on sphere forming ability on HT29, CTC44, Gli4, A549 and Panc-1 in suspension culture. Results are expressed in fold change compared to control and are mean ± SEM of three distinct experiments. ns not significant, *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. One-way Anova followed by multiple comparisons.
<figure><img src="image_65.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>Figure S2: DSM and SM do not affect cell growth or viability in monolayer culture. Related to Figure 2.</figcaption></figure>

A.) DSM and SM do not impact cell growth in monolayer culture. Related to Figure 2. Cell proliferation of SW620, CRC1 and MCF-7 was evaluated in the presence or absence of antibiotics (SM or DSM at 400 µM). Results (expressed in cell index) are the mean ± SEM of a representative experiment (out of three separate experiments). B.) DSM and SM are not toxic in monolayer culture. DSM and SM toxicity were evaluated on SW620, CRC1 and MCF-7 cells. Results are expressed in fold change compared to control (untreated cells) and are mean ± SEM of three distinct experiments.
<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

<figure><img src="image_72.png" /><figcaption>Figure S3: SM-resistant cells deploy a transcriptional anti-oxidative stress response program, related to Figure 4</figcaption></figure>

A.) Gene signature is absent in SM-treated monolayer culture.. SW620 and MCF-7 cells were treated with either SM or DSM at 400 µM in monolayer culture for 4 days. mRNA level was evaluated for mature and immature (unspliced) mRNA. Mean ± SEM of three biological replicates. ns not significant. Two-way Anova followed by multiple comparisons. B.) Western blot analysis showing expression levels of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2). n=3 biological replicates. Mean ± SEM, *p-value < 0.05. C.) mRNA level of NRF2 by qPCR in SW620 and MCF-7 cells. n=3 biological replicates. Mean ± SEM of three biological replicates. ns not significant. Two-sided unpaired T-test. D.) NRF2 knockdown does not affect sphere-forming ability in SW620. Sphere formation assay was performed after siRNA treatment targeting NRF2 in SW620 cell line. Mean ± SEM of three biological replicates. ns not significant. Two-way Anova followed by multiple comparisons. E.) NRF2 knockdown does not affect gene signature monolayer culture of SW620. Mean ± SEM of three biological replicates. ns not significant, **p-value < 0.01. Two-way Anova followed by multiple comparisons
<figure><img src="image_73.png" /><figcaption>**Figure S4:** Cell death inhibitors do not affect sphere forming ability when used in isolation. Related to **Figure 5**. Effects of Z-VAD-FMK (1 µM), CQ (0.5 µM), Y-27632 (10 µM), nec-1 (10 µM) and DFO (1 µM or 2 µM) added in SW620, CRC1 and MCF-7 cultured in suspension culture. Results are expressed in fold change compared to control. n=3 biological replicates. Mean ± SEM, ns not significant. One-way Anova followed by multiple comparisons.</figcaption></figure>
CM-H⁺DCFDA

<figure><img src="image_74.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_75.png" /></figure>

SW620

<figure><img src="image_76.png" /><figcaption>C</figcaption></figure>

CRC1

<figure><img src="image_77.png" /></figure>

MCF-7

<figure><img src="image_78.png" /></figure>

SW620

<figure><img src="image_79.png" /></figure>

Calcein-AM

MCF-7

<figure><img src="image_80.png" /></figure>

SW620

<figure><img src="image_81.png" /><figcaption>E</figcaption></figure>
**Figure S5: Impact of SM on ROS production. Related to Figure 6.**

A.) Total ROS level from SM-treated cells in suspension culture. Flow cytometry quantification of CMH2DCFDA (50 nM SW620, 500 nM MCF-7) mean fluorescence intensity from suspension cultures of either DSM- or SM-treated cells (120 µM for SW620 or 180 µM for MCF-7). Results are expressed in fold change compared to control. n=3 biological replicates. Mean ±SEM, ns not significant, ***p-value < 0.001. Two-sided unpaired T-test. Histogram representative of triplicates. B.) ROS inhibitors restore sphere formation of SM-treated CRC1 cells. SM-treated suspension culture of CRC1 (400 µM) were cotreated with Nac or MT at increasing concentrations. Results are expressed in fold change compared to control. n=3 biological replicates. Mean ±SEM, *p-value < 0.05, ***p-value < 0.001. One-way Anova followed by multiple comparisons. C.) The Superoxide dismutase mimetic Tempol inhibits SM effect on sphere formation. Cells treated with SM at 200 µM (SW620) or 400 µM (MCF-7 and CRC1) were cotreated with Tempol at 100 µM. Results are expressed in fold change compared to control. n=3 biological replicates. Mean ± SEM, ***p-value < 0.001. One-way Anova followed by multiple comparisons. D.) Cytoplasmic iron pool is increased following SM treatment in suspension culture. Flow cytometry quantification of Calcein-AM (10 nM) mean fluorescence intensity in cells cultured in suspension culture with DSM and SM at 120 µM (SW620) or 180 µM (MCF-7). Results are expressed in fold change compared to control. n=3 biological replicates. Mean ± SEM, *p-value < 0.05. Two-sided unpaired T-test. Histogram representative of triplicates. E.) SM treatment does not impact GSH/GSSG ratio in suspension culture. GSH/GSSG ratio was calculated from SM- or DSM-treated (120 µM) SW620 cells grown in suspension for 6 days. Results are expressed in fold change compared to control. n=3 biological replicates. Mean ±SEM, ns not significant. One-way Anova followed by multiple comparisons
B

<figure><img src="image_82.png" /></figure>

C

<figure><img src="image_83.png" /></figure>

D

<figure><img src="image_84.png" /></figure>

<figure><img src="image_85.png" /><figcaption>**Figure S6: SM induces mitochondrial dysfunction. Related to Figure 7.**</figcaption></figure>

A.) mtDNA copy number is not impacted by SM in suspension culture. mtDNA/nucDNA ratio in cells treated with SM and DSM in suspension culture (120 µM SW620, 165 µM CRC1, 180 µM MCF-7). PCR quantification was carried out following RNA extraction. n=3 biological replicates. Mean ± SEM, ns not significant. One-way Anova followed by multiple comparisons. B.) SM treatment does not impact mitochondria morphology in monolayer culture. Ultrastructure of mitochondria from either SM- or DSM-treated monolayer culture of SW620 (400 µM) by TEM. Scale bar 500 nm. C.) mRNA level of MT-CO1 was evaluated for SW620 mRNA. Mean ± SEM of three biological replicates. ns not significant. Two-way Anova followed by multiple comparisons D.) Representative trace of the real-time, high-resolution respirometry experiments. A Substrate-Uncoupler-Inhibitor Titration (SUIT) protocol was used to assess mitochondrial respiratory function. The additions are shown by vertical lines, with each event identified at the top. Omy, Oligomycin; CCCP, Carbonyl cyanide m-chlorophenyl hydrazone, uncoupler; Ama, Antimycin A.
Supplementary Tables

<table><caption>Table S1: List of primers. Related to STAR methods</caption><tr><td>AKR1C2 mature mRNA</td><td>5'-GGGTTCCACCATATTGATTCT-3'</td><td>5'-CACTGCCATCTGCAATCT-3'</td></tr><tr><td>AKR1C2 immature mRNA</td><td>5'-GCCAACGTGAGACAGAGGTG-3'</td><td>5'-AGGTTGACCCATGCTGACCC-3'</td></tr><tr><td>AKR1C3 mature mRNA</td><td>5'-GAGAAGTAAAGCTTTGGAGGTCACA-3'</td><td>5'-CAACCTGCTCCTCATTATTGTATAAATGA-3'</td></tr><tr><td>AKR1C3 immature mRNA</td><td>5'-GGTGGCGATTATGGCTTTGGA-3'</td><td>5'-AGCAGAAGTTGGGAGACCCAG-3'</td></tr><tr><td>HMOX1 mature mRNA</td><td>5'-CCTCCCTGTACCACATCTAT-3'</td><td>5'-AGCTCTTCTGGGAAGTAGAC-3'</td></tr><tr><td>HMOX1 immature mRNA</td><td>5'-GGCACTGTGGCCTTGGTCTA-3'</td><td>5'-ACTTCTCCTACCGAGCACGC-3'</td></tr><tr><td>OSGIN1 mature mRNA</td><td>5'-CTTCTACGCCAGACACAGAC-3'</td><td>5'-GGATCACCATGGAGCCTTC-3'</td></tr><tr><td>OSGIN1 immature mRNA</td><td>5'-GGTCCCAAATAAAGCCAGTGCC-3'</td><td>5'-TGGACCTCATCCAGGACCACA-3'</td></tr><tr><td>UGDH mature mRNA</td><td>5'-CAACAGCGATTGGAATGGACC-3'</td><td>5'-TCTGGCAAATTCAGAGCCTCA-3'</td></tr><tr><td>UGDH immature mRNA</td><td>5'-TGCCCATCGTTAGCTCCTGG-3'</td><td>5'-CCAACCCAGAAGGGGTCACT-3'</td></tr><tr><td>SQSTM1 mature mRNA</td><td>5'-AGGCGCACTACCGCGAT-3'</td><td>5'-CGTCACTGGAAAAGGCAACC-3'</td></tr><tr><td>SQSTM1 immature mRNA</td><td>5'-GCCAGCTCAGCTTGTACTCA-3'</td><td>5'-CCCTCAAATTGCTGACCCCT-3'</td></tr><tr><td>GPX2 mature mRNA</td><td>5'-GCCTCCTTAAAGTTGCCATA-3'</td><td>5'-GCCCAGAGCTTACCCA-3'</td></tr><tr><td>GPX2 immature mRNA</td><td>5'-GTGCAATTGAAAGCAGGCGA-3'</td><td>5'-CTACCGACCCACCTACCCAT-3'</td></tr><tr><td>TXNRD1 mature mRNA</td><td>5'-GTTACTTGGGCATCCCTGGTGA-3'</td><td>5'-CGCACTCCAAAGCGACATAGGA-3'</td></tr><tr><td>TXNRD1 immature mRNA</td><td>5'-AACCGTGGACAGAATTGTCATT-3'</td><td>5'-GCTGAGGGCAATGTTCTAATGT-3'</td></tr><tr><td>Oct4</td><td>5'-GTGGAGAGCAACTCCGATG-3'</td><td>5'-TGCTCCAGCTTCTCCTTCTC-3'</td></tr><tr><td>CD44</td><td>5'-TGGCACCCGCTATGTCGAG-3'</td><td>5'-GTAGCAGGGATTCTGTCTG-3'</td></tr><tr><td>CD133</td><td>5'-TGGGGCTGCTGTTTATTATTCT-3'</td><td>5'-TGCCACAAAACCATAGAAGATG-3'</td></tr><tr><td>CD26</td><td>5'-CAAATTGAAGCAGCCAGACA-3'</td><td>5'-CACACTTGAACACGCCACTT-3'</td></tr><tr><td>GAPDH</td><td>5'-CCCACTCCTCCACCTTTGAC-3'</td><td>5'-CCACCACCCTGTTGCTGTAG-3'</td></tr><tr><td>Actin</td><td>5'-AGCACGGCATCGTCACCAACT-3'</td><td>5'-TGGCTGGGGTGTTGAAGGTCT-3'</td></tr><tr><td>MT- CO1</td><td>5'-CTAGCAGGTGTCTCCTCTATCT-3'</td><td>5'-GAGAAGTAGGACTGCTGTGATTAG-3'</td></tr><tr><td>β2-microglobulin</td><td>5'-TGCTGTCTCCATGTTTGATGTATCT-3'</td><td>5'-TCTCTGCTCCCCACCTCTAAGT-3'</td></tr><tr><td>Universal forward</td><td>5'-TTAACTCCACCATTAGCACC-3'</td><td>5'-GAGGATGGTGGTCAAGGGA-3'</td></tr></table>