Data and metadata supporting the published article: Immunostimulatory and anti-tumor metronomic cyclophosphamide regimens assessed in primary orthotopic and metastatic murine breast cancer

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).

Data access: 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 (https://doi.org/10.6084/m9.figshare.12383498). Datasets supporting the supplementary figures in the published article, are available on reasonable request from the corresponding authors Dr. Kabir Khan, email address: kkhan@sri.utoronto.ca, and Dr Robert Kerbel, email address robert.kerbel@sri.utoronto.ca.

Study approval: Animal experiments were carried out with the approval of the institutional Animal Care Committee in accordance to Canadian Council on Animal Care (CCAC) guidelines.

Study aims and methodology: 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?

For in vitro 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: in vivo therapies, postsurgical metastasis studies, 4H-CTX in vitro studies, flow cytometry, CyTOF acquisition and analysis, immunohistochemistry, and statistical analysis.

Dataset descriptions:

The datasets generated during this study are Figure_1.xlsx, Figure_2.xlsx, Figure_3.xlsx, Figure_4.xlsx, Figure_5.xlsx and Figure_6.xlsx are in .xlsx file format, and support figures 1, 2, 3, 4, 5 and 6 respectively.

This data record also includes the following datasets in .fcs file format: YS20170831_Control.fcs, YS20170831_CTX1401q6d.fcs, YS20170831_LDM_CTX.fcs and YS20170831_MTD_CTX.fcs. The .fcs file include the CyTOF data, and support figure 3C.

The rar zip file Khan_et_al_2020_NPJBC_supplementarydata.rar 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.

Figure_1.xlsx: 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.

Figure_2.xlsx: 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.

Figure_3.xlsx: 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.

Figure_4.xlsx: 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.

Figure_5.xlsx: 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.

Figure_6.xlsx: 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.

Software needed to access data: 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.