Chemotherapeutic drugs cause increased release of pro-inflammatory molecules from MM cells.

<p>(A) Hmeso MM cells treated with either Dox or cisplatin caused increased release of HMGB1, FGF2, IL-1β and IL-18 as measured by Western blot analysis or ELISA. No loading controls were included as analysis was performed in cell culture medium. (B) H2373 MM cells also showed increased release of various pro-inflammatory molecules in response to drugs. IL-6 levels were either decreased (H2373) or not significantly changed (Hmeso) by drug treatments. *p≤0.05 as compared to untreated controls (0).</p

MM cells and tumors have low levels of NLRP3 protein and caspase-1 activity.

<p>A. Seven human MM cell lines were assessed for NLRP3 steady-state mRNA levels and compared with human mesothelial cell line (LP9), *p≤0.05 as compared to MM cell lines; B. NLRP3 protein levels in 4 human MM cell lines as compared to LP9 (red-NLRP3 protein, green-nucleus); C. NLRP3 mRNA levels in human MM tumor tissues (T) and normal counter parts (N); D. NLRP3 protein levels in human MM tumor tissue arrays as compared to normal lung (red-NLRP3 protein, green-nucleus); E. Caspase-1 activity in human MM cell lines as compared to LP9, *p≤0.05 as compared to MM cell lines. Scale bar = 50 microns.</p

Combination of IL-1R antagonist and cisplatin attenuated MM tumor weight/volume in an intraperitoneal xenograft mouse model.

<p>SCID mice injected with Hmeso MM cells received either cisplatin (2mg/kg, ip, 1X/week for 2 weeks), Anakinra (92 mg/kg, ip, 2x daily for 3 weeks), Cisplatin and Anakinra together or saline. (A, B) tumor weights and volumes were drastically reduced in combination group. (C) Total cell count was not significantly affected by combination treatment. (D) Neutrophil counts were significantly lower in cisplatin and combination group. *p≤0.05 as compared to saline treated group; †p≤0.05 as compared to cisplatin alone (by Student’s‘t’ test).</p

Chemotherapeutic drugs cause pyroptosis in MM cell lines.

<p>MTS assay performed to measure cell viability on Hmeso or H2373 MM cells in presence of Dox or cisplatin with and without specific caspase-1 inhibitor (A, B) or pan caspase inhibitor (C, D). The effect of these inhibitors on drug-induced caspase-1 activity was also performed in both MM cell lines (E, F). *p≤0.05 as compared to untreated controls (0); †p≤0.05 as compared to similar treatment without caspase- inhibitor, ‡ p≤0.05 as compared to low dose caspase inhibitor.</p

Chemotherapeutic drugs cause priming and activation of the NLRP3 inflammasome in MM cell lines.

<p>Treatment of Hmeso MM cells with Dox or cisplatin resulted in increased NLRP3 protein levels (A) and activation of inflammasome (B). Activation was measured by increased caspase-1 p20 release into the medium. *p≤0.05 as compared to untreated controls (0). (C) Cisplatin increased the protein levels of NLRP3 in H2373 MM cells and both Dox and cisplatin caused activation of caspase-1 in H2373 cells as measured by caspase-1 p20 release into the medium (D). *p≤0.05 as compared to untreated controls.</p

Chemotherapeutic drugs increase NLRP3 levels in human MM cell lines.

<p>Human MM cell lines were treated with different concentrations (0–100 μM) of cisplatin or Dox (0–25 μM,) for 24 or 48 h and steady-state NLRP3 mRNA levels were assessed by qRT-PCR. *p≤0.05 as compared to untreated (0) controls at the same time point.</p

Chemotherapeutic drugs regulate PYCARD/ASC and pro-caspase levels in MM cell lines.

<p>Human MM cell lines were treated with different concentrations (0–100 μM) of cisplatin or Dox (0–25 μM) for 24 or 48 h and steady-state PYCARD/ASC or pro-caspase-1 mRNA levels were assessed by qRT-PCR. *p≤0.05 as compared to untreated (0) controls at the same time point.</p

S3 Data -

The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFβ pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.</div

Single cell analysis of patient-derived MSCs.

A) CD90+ cells were isolated from normal donors or patients with invasive breast cancer and subjected to single cell analysis. Gene expression profiles from single cells were clustered using tSNE, and 7 distinct cell clusters were observed. B) Candidate gene expression profiles were used to functionally characterize MSCs into 3 main subclasses (osteogenic, chondrogenic or adipogenic). C) Comparison of cells derived from healthy donors or breast cancer patients demonstrated proportional changes in number of cells contributing to specific clusters. D) Ontology categories associated with single cell populations.</p

Schematic of sample acquisition for analysis.

A) CD90 positive mesenchymal stromal cells isolated from breast cancer patients express a unique gene expression profile indicative of increased cell to cell signaling. MSCs were isolated from fresh stromal tissue adjacent to an invasive lesion (tumor-associated) or distal to the tumor site (patient-normal). B) Microscopy images demonstrating phenotypic characteristics of CD45+ or CD45-/CD90+ cells isolated by magnetic-activated cell sorting (MACS). C) CD90+ cells were subjected to FACS for phenotypic mesenchymal associated cell surface markers. D) Microscopy images demonstrating tumor tissues used for FFPE gene expression analysis. Hematoxylin and Eosin staining of tissue sections used for analysis (left panel) and images of tumor sections before and after Laser Capture microdissection (LCM) (right panels).</p