Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways

Abstract

Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.

Figure 1. Stromal cells induce ISGs and protect basal-like breast cancer cells against radiation in a STAT1-dependent manner

A) Human MDA-MB-231 metastatic breast cancer cell (BrCa) line (1833) was admixed with or without MRC5 normal human fibroblasts (Stroma) and expression of IRDS genes was determined by qRT-PCR. B) GFP-labeled 1833 breast cancer cells with and without MRC5 fibroblasts were xenografted subcutaneously into nude mice and tumors imaged (20X) at day 14. STAT1 intensity in breast cancer cells is quantitated for representative field shown. Scale bar is 100 microns. C) Bioluminescence imaging (BLI) response of 1833 breast cancer cells with a luciferase reporter gene after xenografting with and without MRC5 fibroblasts. Tumors were irradiated with 8 Gy (day 0). Shown is change in photon flux over time (first derivative, mean ± SEM, n=5–10). Positive first derivative indicates growth, zero indicates no growth, and negative values denote regression. Data are a separate analysis of the control groups from Figure 5M. D) 1833 breast cancer cells were stained with GFP and TUNEL (red) 10 days after RT. Percent TUNEL positive is shown. Scale bar is 100 microns. E) Breast cancer cells (Table S1) were classified as IRDS responders (IRDS-Rs) or IRDS non-responders (IRDS-NRs). Heat map and scale shows breast cancer IRDS genes after mono-culture (M) or MRC5 co-culture (C). F) Cell death of IRDS-Rs and IRDS-NRs four days after 10 Gy RT in mono- (Mono) and co-culture (Co-cx) (n=3–10). G) Cell death of 1833 IRDS-R after cisplatin chemotherapy (n=3) and after dose response. H) Gene Set Analysis shows changes in IRDS genes 48 hrs after co-culture vs mono-culture of IRDS-Rs (left, also see Table S1), or after STAT1 knockdown in 1833 IRDS-R in co-culture (right). Top graph plots individual and overall gene scores, and bottom graph shows fold-change. I) Cell death of 1833 IRDS-R four days after 10 Gy RT using three independent siRNAs to STAT1. J) BLI-based survival assay after 10 Gy RT (day 0) using luciferase-labeled 1833 cells with shSTAT1 or control knockdown (shCont). Photon flux (×10⁶) for each well is indicated. Shown is representative experiment (n=5). *p < 0.05. See Figure S1.

Figure 2. Stromal cell interaction increases exosomes that upregulate ISGs through a RIG-I anti-viral pathway

A) Importance scores (higher is more predictive) of PRRs from a multivariable random forest (RF) regression model to predict induction of IRDS after MRC5 co-culture with IRDS-Rs. The model explains 60.8% of the total variance. Adjusted effect of RIG-I on IRDS metagene expression is shown on right (red dashes are ± two SE). B) Expression of IRDS genes after siRNA to RIG-I (top row) or MYD88 (bottom row) in 1833 IRDS-R. Shown is a representative experiment (n=3). C) Cell death of 1833 IRDS-R after RT (n=4) and a representative BLI-based survival assay (n=2) after the indicated knockdown (RT on day 0). Photon flux (×10⁶) for each well is shown. The control is same as Fig. 1J. D) Expression of IRDS genes in 1833 IRDS-R (middle) or MCF7 IRDS-NR (right) after addition of conditioned media (CM) from MRC5 fibroblasts (Stroma), IRDS-R or IRDS-NR (BrCa), or MRC5 co-culture with IRDS-Rs or IRDS-NRs (Co-cx). See schematic (left). E) CM collected after 48 hrs or the soluble fraction from CM (Soluble) was applied to 1833 IRDS-R and expression of IRDS genes was examined (n=4). F) Fold induction of IRDS genes in 1833 IRDS-R after addition of co-culture CM or purified exosomes (n=5). G) NanoSight quantification of exosomes (left) from 1833 IRDS-R, MRC5 fibroblasts (Stroma), and MRC5 co-culture using either 1833 IRDS-R or IRDS-NR (MDA-MB-468 or MCF7). Immunoblot for TSG101 (right) using 1833 IRDS-NR and MDA-MB-468 IRDS-NR. H) MRC5 fibroblasts (Stroma) or 1833 IRDS-R were labeled with green or red lipophilic dye in mono-culture (left and middle). For co-culture (right), MRC5 (arrows) were labeled red and breast cancer cells green. Scale bar is 40 microns. I) Representative flow cytometry of DiD dye transfer from MRC5 stroma to 1833 IRDS-R or MDA-MB-468 IRDS-NR. J) Exosome transfer from co-culture after TSG101 knockdown (left) and after addition of the co-culture CM cleared of debris and apoptotic bodies (right) (n=4). K) IRDS gene induction by co-culture CM after TSG101 knockdown in 1833 IRDS-R, MRC5 stroma, or both (n=3). Gene expression and significance levels are relative to siControl. *p < 0.05. See Figure S2.

Figure 3. Stromal exosomes are regulated by RAB27B and transfer 5’-triphosphate RNA to activate RIG-I in breast cancer cells

A) Exosomes were isolated from mono-culture of MRC5 fibroblasts (Stroma) or 1833 IRDS-R (right) or from co-culture (left) and profiled by antibody array for the indicated exosome markers. GM130 is a check for cellular contamination. Positive (+) and negative (−) controls are labeled. B) Averaged microarray expression of the indicated RABs from MRC5 in mono-culture (Stroma) or after co-culture with IRDS-R or IRDS-NR are shown as a heat map. Immunoblot (right) for Rab27b protein expression in MRC5 after co-culture with MDA-MB-157 or 1833 IRDS-R (Figure S3A) compared to MRC5 mono-culture. C) IRDS expression in 1833 IRDS-R after addition of CM isolated from co-culture using MRC5 transfected with siRAB27B compared to siControl (n=3). D) Exosome transfer to 1833 IRDS-R after co-culture with or without RAB27B knockdown (left) or addition of co-culture CM cleared of debris and apoptotic bodies (right). E) Average IRDS gene expression (mean expression of IFIT1, MX1, and STAT1) in response to exosomes (Exo, n=5) or co-culture CM (n=6) plotted against RIG-I levels after knockdown in 1833 IRDS-R. F) IRDS gene expression from two representative data points used to generate plot in Figure 3E are shown relative to siControl. G) IRDS gene expression after RNA from exosomes (ExoRNA), cellular RNA, or a positive control HCV RNA was transfected into 1833 IRDS-R with or without RIG-I knockdown (n=4). IFI16 is a non-IRDS gene used as a negative control. H) Expression of IRDS genes IFIT1 and MX1 resulting from transfection of ExoRNA after RNase treatment, or I) removal of 5’-monophosphate (5’-p) and/or 5’-triphosphate (5’-ppp) (n=3). An in vitro transcribed 5’-ppp RNA (IVT5’ppp) is used as a positive control. Shown are RNA motifs remaining after enzyme modification with alkaline phosphatase (AlkPase), Terminator exonuclease (Term), and tobacco acid pyrophosphatase (TAP). IVT5’ppp serves as a control for RNA enzyme modification by AlkPase and TAP. J) Distribution of known gene transcripts and intergenic transcripts from rRNA-depleted exoRNA and cellular RNA from 1833 IRDS-R co-culture (left). Distribution of major repetitive elements and transposable element classes for intergenic transcripts are shown on right. K) ExoRNA enrichment for major subfamilies of transposable elements and satellite sequences compared to cellular RNA. *p < 0.05. See Figure S3.

Figure 4. STAT1 enhances the transcriptional response to juxtacrine NOTCH3 signaling that is required for stroma-mediated protection

A) Cell death of 1833 IRDS-R in co-culture after RT. MRC5 fibroblasts were separated by a transwell filter large enough to allow exosome passage (n=3). B) Immunoblot of the indicated NOTCH family members in 1833 IRDS-R after mono-culture (M) or co-culture (C). Arrow indicates cleaved intracellular domain. C) Expression of NOTCH target genes in IRDS-R and IRDS-NR after co-culture, and D) after STAT1 knockdown in 1833 IRDS-R after co-culture. Notch targets were experimentally defined by GSI washout (Table S4) and used in Gene Set Analysis. E) Expression of the indicated NOTCH target gene primary transcript (PT) in 1833 IRDS-R (n=3). F) Expression of HEY1 PT in response to doxcycyline (Dox) induced NICD3 in 1833 IRDS-R with or without addition of co-culture CM (mean ± SEM, n=6–8). Inset shows NICD3 levels after Dox addition (µg/ml). G) Expression of the indicated primary transcripts to NICD3 after addition of co-culture CM or CM depleted of exosomes (Exo dep). CM compared to CM depleted of exosomes is used for significance levels (mean ± SEM, n=4–6). H) ENCODE ChIP data for STAT1 occupancy of the HEY1 proximal promoter region is shown along the indicated genomic coordinates. Bar plots show STAT1 ChIP from 1833 IRDS-R with and without addition of CM (left) and after mono- or co-culture (right). Relative position upstream of the transcriptional start site (TSS) is labeled on the x-axis for each bar plot. Shown are two representative experiments (mean ± SD) out of four total. I) Expression of HEY1 and HES1 mRNA or primary transcripts in response to NICD3 and co-culture CM in 1833 IRDS-R with and without STAT1 knockdown (mean ± SEM, n=4–7). ●p<0.10, *p < 0.05. See Figure S4.

Figure 5. Stromal cells drive the expansion of a subpopulation of therapy resistant breast cancer cells through anti-viral STAT1 and NOTCH3 signaling

A) Gene Set Analysis comparing IRDS-R in mono-culture versus co-culture with MRC5 fibroblasts, or comparing 1833 IRDS-R in co-culture transfected with siSTAT1 vs. siControl. B) Percentage of CD44⁺CD24^low+ 1833 IRDS-R after co-culture with MRC5. All CD24^low+ cells are also CD44⁺. C) Survival of sorted CD44⁺CD24^low+ and CD44⁺CD24^neg cells after 10 Gy RT or 4 µM doxorubicin (chemo). Number of mammospheres from 1833 IRDS-R after D) co-culture, or E) co-culture following knockdown of STAT1 (siS1), NOTCH3 (siN3), or control (siCt), or after treatment with the GSI DAPT. F) Number of mammospheres after NICD3 induction by doxycycline in mono-culture. G) Proportion of surviving mammospheres relative to untreated control in mono- or co-culture after 3 Gy RT. Cell death after 10 Gy RT following H) knockdown of NOTCH3 in 1833 IRDS-R, I) knockdown of JAG1 in 1833 IRDS-R, MRC5 (Stroma), or both (n=4), J) expression of NICD3 (n=7), or K) STAT1 knockdown with and without NICD3 expression (n=3–4). L) Cell death of IRDS-Rs and IRDS-NRs after 10 Gy RT and treatment with the GSI DAPT or DMSO (n=5–10). M) Photon flux from mice xenografted subcutaneously with luciferase-labeled 1833 IRDS-R with or without MRC5 fibroblasts (Stroma) and treated 7 days later with 8 Gy RT, the GSI DAPT, both, or untreated. Mean values (black “X”) are connected by blue line. Representative tumors after treatment are inset. In presence of stroma, tumor response was associated with RT (p < 0.001) and GSI (p=0.004). Without stroma, RT (p=0.019) but not GSI (p=0.79) was associated with response. N) Percentage of CD44⁺CD24^low+ cells in tumors from mice xenografted with 1833 IRDS-R with and without MRC5 stroma 7 days after the indicated treatment. O) Survival of these mice, which are independent cohorts from that used in Fig. 5M. *p < 0.05. See Figure S5.

Figure 6. Expression of anti-viral and NOTCH3 pathway predict IRDS and NOTCH target gene expression in primary human and mouse tumors

A) Expression of RAB27B, STAT1, and NOTCH3 in primary human triple negative breast cancer (TNBC), or B) in TNBC patient-derived xenografts (PDX) and basal-like tumors from K14Cre;BRCA1^F/F;p53^F/F conditional knockout mice. Arrows show representative areas of stroma. Insets for TNBC images show darker staining regions (red) segmented from lighter regions. Semi-quantitation of expression in stroma (S), tumor (T), or tumor-stroma borders (B) is indicated. Vertical bar is 200 microns. A total of seven primary TNBC tumors were scored. Two out of 2 PDX and 3 out of 3 mouse tumors gave similar results. Shown are representative images and semi-quantitation. C) Box- and-whisker plots of expression values for the indicated RABs from primary human breast cancer stroma (Tumor) or normal stroma (Norm) using the Stroma series. D) Importance scores (higher is more predictive) from a RF regression model (variance explained: 55.1%) to predict breast cancer IRDS expression using the NKI295 series. Adjusted effect of RIG-I on IRDS expression (right). E) Heat map and scale showing expression of all available NOTCH receptors in breast cancer (brown) and NOTCH ligands in stroma (green) from the LCMD series. These were used to predict the average expression of NOTCH target genes in breast cancer (variance explained: 30.2 ± 1.1%) defined by GSI washout (NOTCH Meta). On the right are importance scores from Monte Carlo replications. See Figure S6.

Figure 7. NOTCH3 and STAT1/IRDS cooperate to predict NOTCH target genes and clinical resistance to chemotherapy and RT preferentially in basal-like breast cancers

Prediction of NOTCH target gene expression by IRDS and NOTCH3/JAG1 in A) primary human tumors and in B) basal-like tumors from the K14Cre;BRCA1^F/F;p53^F/F conditional knockout mice. For human tumor analysis, the NKI295 series was used. The probability of NOTCH pathway activation as measured by the NOTCH metagene is shown on the y-axis with probabilities for basal (red dots) or non-basal (blue dots) tumors displayed separately. The percentage of tumors with greater than 80% probability of NOTCH activation is inset. A LOWESS regression line (black dashed line) is shown. IRDS and JAG1 were equally divided into low, intermediate, and high values. For mouse tumor analysis, IRDS, NOTCH3, and JAG1 expression were dichotomized into only high and low due to smaller sample size. Mean value is marked by red line. C) Heat map showing probabilities of NOTCH activation and NOTCH3 expression for each patient (columns) in the NKI295 series. All values are scaled between 0 and 1. Hatches below the heat map show status for IRDS(+), NOTCH3(hi), and the indicated molecular subtypes. On the right is Gene Set Analysis for the same TIC signature used in Fig. 5A and compares NOTCH3(hi)/IRDS(+) tumors to those that are NOTCH3(lo) and/or IRDS(−). D) Survival after adjuvant chemotherapy of patients from the NKI295 series stratified by NOTCH3 and IRDS. Overall p-value is shown. E) Hazard ratios and 95% confidence intervals from Cox regression analysis for breast cancer survival using NOTCH3 as a continuous variable, IRDS status (positive vs. negative), and MammaPrint (Mamma) metastasis signature status (positive vs. negative). All patients received adjuvant chemotherapy. Hazard ratio for NOTCH3 is per unit increase in expression. Analyses are also stratified by IRDS status and basal vs. non-basal subtype tumors. Values are not shown if there are too few patients in the group. F) Relapse in irradiated region (local-regional control) after adjuvant RT. G) Hazard ratio from Cox regression for relapse in the Stroma series using stromal RAB27B as a continuous variable. H) Model of the tumor-stroma anti-viral/NOTH3 pathways controlling RT/chemo resistance. See Figure S7.