DOI: 10.6084/m9.figshare.12383498.v1
title: Data and metadata supporting the published article: Immunostimulatory and anti-tumor metronomic cyclophosphamide regimens assessed in primary orthotopic and metastatic murine breast cancer

description: The primary goal of this study was to comparatively evaluate three different cyclophosphamide (CTX) protocols for anti-tumor efficacy in three highly translatable murine breast cancer models with particular emphasis on an every 6 day metronomic-like regimen using a dose of 140 mg/kg (CTX1401q6d).<br><b>Data access</b>: All the datasets supporting figures 1-6 in the published article, are publicly available in the figshare repository as part of this figshare data record (<b>https://doi.org/10.6084/m9.figshare.12383498</b>). Datasets supporting the supplementary figures in the published article, are available on reasonable request from the corresponding authors Dr. Kabir Khan, email address: <b>kkhan@sri.utoronto.ca, </b>and Dr Robert Kerbel, email address <b>robert.kerbel@sri.utoronto.ca.</b><br><b>Study approval</b>: Animal experiments were carried out with the approval of the institutional Animal Care Committee in accordance to Canadian Council on Animal Care (CCAC) guidelines.<br><b>Study aims and methodology</b>: The purpose of this study was to address three related questions. First, what is the comparative effectiveness of three different CTX regimens, one being a maximum tolerated dose (MTD) protocol, a second being low dose daily/continuous oral metronomic CTX, and the third being the CTX140 1q6d regimen. Second, what is the evidence for immunological mechanisms mediating anti-tumor efficacy of the CTX140 1q6d regimen? Third, would concomitant use of the immune system boosting CTX140 1q6d regimen improve outcomes of PD-L1 antibody therapy?<br>For <i>in vitro</i> experiments, the following cell lines were used: EMT6/P (EMT6-parental), EMT6-CDDP (EMT6-cisplatin resistant) and SP1-AC2M2.For the animal experiments, female BALB/c, CBA/J, and SCID CB-17 mice were purchased from Jackson Laboratories. Female SCID Beige (CB17.Cg- rkdcscidLystbg-J/Crl) mice were purchased from Charles River Laboratories.The following techniques are described in more detail in the published article: <i>in vivo</i> therapies, postsurgical metastasis studies, 4H-CTX <i>in vitro</i> studies, flow cytometry, CyTOF acquisition and analysis, immunohistochemistry, and statistical analysis.<b><br></b><b>Dataset descriptions:</b>The datasets generated during this study are <b>Figure_1.xlsx, Figure_2.xlsx, Figure_3.xlsx, Figure_4.xlsx, Figure_5.xlsx </b>and <b>Figure_6.xlsx</b> are in <b>.xlsx</b> file format, and support figures 1, 2, 3, 4, 5 and 6 respectively.This data record also includes the following datasets in <b>.fcs </b>file format: <b>YS20170831_Control.fcs, YS20170831_CTX1401q6d.fcs, YS20170831_LDM_CTX.fcs </b>and<b> YS20170831_MTD_CTX.fcs</b>. The .fcs file include the CyTOF data, and support figure 3C.The rar zip file <b>Khan_et_al_2020_NPJBC_supplementarydata.rar </b>supports supplementary figures 1-8 in the published article. These files will be made available on request as described in the Data access section above.<br><br><b>Figure_1.xlsx</b>: a Raw tumor volumes of mice bearing EMT6/P treated with either saline control, MTD CTX, LDM CTX continuous daily or CTX140 1q6d. b Endpoint data from Figure 1a for survival analyses. C Raw tumor volumes of SCID Beige mice bearing EMT6/P treated with either saline control, MTD CTX, LDM CTX continuous daily or CTX140 1q6d. d Endpoint data from Figure 1c. e Tumor volume comparison at 32 days post implantation in BALB/c wild type, SCID and SCID Beige mice all treated with CTX140 1q6d. Percentage change of mouse weights from 1a also included.<b>Figure_2.xlsx</b>: a Raw tumor volume data of CBA/J mice bearing SP1-AC2M2 tumors treated with either saline control, MTD CTX, LDM CTX continuous or CTX140 1q6d. b Survival data from Figure 2a when mice reached tumor endpoint as a surrogate for survival. c Raw tumor volume data of mice displaying complete responses that were re-challenged with SP1-AC2M2 in the opposite mammary fat pad to the original implantation. d Raw tumor volumes of SP1-AC2M2 primary tumors were treated with CTX140 1q6d (blue arrows), a break was initiated after 5 doses. Percentage change of mouse weights from 2a also included.<b>Figure_3.xlsx</b>: a Raw tumor volumes of mice bearing EMT6-CDDP treated with either saline control, MTD CTX, LDM CTX continuous daily or CTX140 1q6d. b Endpoint survival data from Figure 3a. Percentage change of mouse weights from 3a also included.<b>Figure_4.xlsx</b>: b Endpoint survival data from EMT6-CDDP adjuvant treatment experiment. c Percentage values of lung metastasis visualised by immunohistochemical staining using Ki67 as a marker for proliferation. Percentage change of mouse weights from 4b also included.<b>Figure_5.xlsx</b>: b Adjusted mean values of fluorescence of EMT6-CDDP, c EMT6/P, or d SP1-AC2M2 cells treated with 20 µM 4-HCTX or 1 µM of PTX, values represent mean fluorescence intensity of PD-L1 analysed by flow cytometry with subtraction of mean fluorescence intensity of isotype IgG.<b>Figure_6.xlsx</b>: a Raw tumor volumes of EMT6-CDDP treated concomitantly with either IgG isotype control or PD-L1 (6E11) blockade in combination with each regimen of CTX or saline control. b Endpoint survival data from Figure 6a. c Raw tumor volumes from induction therapy with CTX140 1q6d then followed up with either IgG isotype or anti PD-L1 in EMT6-CDDP model. Percentage change of mouse weights from 6a also included.<br><b>Software needed to access data</b>: The flow cytometry files in .fcs file format can be accessed and analysed using a software such as FloJo. The files are fully annotated for which channels refer to which cell markers were used.<br>