Neoplastic Immune Mimicry Potentiates Breast Tumor Progression

Abstract

Dedifferentiation programs are commonly enacted during breast cancer progression to enhance tumor cell fitness. Increased cellular plasticity within the neoplastic compartment of tumors correlates with disease aggressiveness, often culminating in greater resistance to cytotoxic therapies or augmented metastatic potential. In this study, we found that subpopulations of dedifferentiated neoplastic breast epithelial cells express canonical leukocyte cell surface receptor proteins and have thus named this cellular program "immune mimicry." Analysis of public human breast tumor single-cell RNA sequencing datasets and histopathologic breast tumor specimens, as well as functional experiments in vitro in breast cancer cell lines and in vivo in murine transgenic and cell line-derived mammary cancer models, showed that neoplastic cells engaged in immune mimicry. Immune-mimicked neoplastic cells harbored hallmarks of dedifferentiation and were enriched in treatment-resistant and high-grade breast tumors. In aggressive breast cancer cell lines, antiproliferative cytotoxic chemotherapies drove epithelial cells toward immune mimicry. The expression of the CD69 leukocyte activation protein by neoplastic cells conferred a proliferative advantage that facilitated early tumor growth. Together, these findings suggest that neoplastic breast epithelial cells upregulating leukocyte surface receptors potentiate malignancy and that neoplastic immune mimicry has potential clinical utility for patient prognosis and stratification.

Significance: A subset of neoplastic breast epithelial cells express surface receptors canonically attributed to leukocytes and are associated with therapy resistance and aggressive tumor behavior.

Figure 1.. Neoplastic immune mimicry is identified in independent human breast tumor scRNA-seq cohorts.

(A) scRNA-seq workflow to collect neoplastic cells based on mammary keratin gene expression. (B) Example of scRNA-seq clustering from the Pal et al. dataset where cells are colored according to their patient identity. Indicated clusters depict neoplastic cell states shared across patients that (C) express myeloid-like, lymphoid-like, neural-like, and mesenchymal-like gene signatures. (D) Immune mimicry is signified by neoplastic cells expressing canonical leukocyte surface receptor genes in a concordant manner across distinct scRNA-seq datasets. (E) The vast majority of breast tumors analyzed have neoplastic cells expressing these immune mimicry markers as a minority subpopulation. (F) Neoplastic cells found within immune-like scRNA-seq clusters are enriched for dedifferentiated, NFκB pathway, and proliferative gene signatures. (G) Neoplastic immune mimicry is elevated in high-grade breast tumors. Breast tumors grade 1–2, n = 22; grade 3, n = 44; tumors with both pre- and post-treatment data depicted once. (H) Neoplastic cells expressing the CD69 early activation marker are increased in tumors subjected to therapy before surgical resection, (I) in scRNA-seq samples harboring a smaller epithelial compartment, and (J) they are more often classified as mitotic. In (H), therapy-naïve tumors, n = 59; treated tumors, n = 10. In (I), 69 breast tumors shown. In (J), 58 tumors with at least 10 CD69-pos neoplastic cells depicted. * p < 0.05; ** p < 0.01; **** p < 0.0001. Wilcoxon signed-rank test (F), Mann-Whitney test [(G) and (H)], Pearson correlation (I) or two-way ANOVA with Šidák correction (J). Data are means ± SEM.

Figure 2.. The immune mimicry CD69 biomarker is associated with aggressive breast cancer in histopathological samples.

(A) Hematoxylin and Eosin (H&E) staining of human breast tumors. These same tissues were subsequently subjected to multiplexed immunohistochemistry to detect epithelial CD69. Scale bar, 200 μm. (B) Multiplexed immunohistochemistry examples of neoplastic CD69 or the lack thereof in primary human breast tumors. Neoplastic CD69 is denoted by overlapping PANCK (red) and CD69 (yellow). The first example portrays a lack of neoplastic CD69 amid immune cell (CD3-pos, cyan) expression. The second and third examples show neoplastic CD69 in diffuse and concentrated staining patterns, respectively. Scale bar, 200 μm. The insets provide magnified views of CD69 (left) versus CD3 and pan-cytokeratin (right). Inset scale bar, 50 μm. (C) Numerous human breast tumors have detectable levels of neoplastic CD69. Plot depicts the percent of epithelial cells strongly positive for CD69 for distinct tumors along with clinical parameters. Epithelial CD69 is summarized from a conservative gating approach applied to multiple regions in fifty-eight tumors.

Figure 3.. Breast cancer cell lines express leukocyte surface receptor RNA and protein.

(A) Interrogation of immune mimicry gene expression in public bulk RNA-seq DepMap data encompassing fifty-seven breast cancer cell lines. (B) Neoplastic immune mimicry RNA gene expression is highest in breast cancer cell lines of the basal phenotype. Breast cancer cell lines expressing greater levels of leukocyte surface receptor genes positively correlate with (C) mammary stem, (D) mammary luminal progenitor, and (E) NFκB pathway gene signatures. (F) Example of flow cytometric CD45 protein staining in MDA-MB-231 cells. Negative (fluorescence minus one) control shown for comparison. (G) Demonstration of canonical leukocyte surface receptor protein positivity in ten human breast cancer cell lines via flow cytometry. Data are monostains performed in biological triplicate. (H) Triple-negative breast cancer cell lines exhibit greater levels of leukocyte surface receptor protein. (I) Immune-mimicked CD45-pos cells are significantly smaller than CD45-neg cells. (J) Immune mimicry marker correlations across the ten cell lines subjected to flow cytometry analysis. Coefficients for significant correlations are indicated. ** p < 0.01; **** p < 0.0001. One-way ANOVA with Tukey correction [(B) and (H)], Pearson correlation [(C), (D), (E) and (J)], or Wilcoxon signed-rank test (I). Data are means ± SEM.

Figure 4.. Cytotoxic therapy further induces neoplastic immune mimicry and CD69.

(A) Cell stress stimuli robustly expand the subpopulation of cells expressing CD45 and CD69 in MDA-MB-231. Scale bar, 50 μm. (B) Example of immune surface receptor positivity for MDA-MB-231 cells across various cytotoxic conditions. Data represent flow cytometric analysis of all stains performed in biological triplicate. (C) CD45 and CD69 are induced at the RNA and protein level in MDA-MB-231 in response to paclitaxel. Data reflect five biological replicates with surface protein measured as flow cytometry monostains. (D) Flow cytometric example of paclitaxel treatment expanding the CD69-pos subpopulation in murine mammary 4T1 cells. Scale bar, 25 μm. (E) CD45 and CD69 are transcriptionally elevated in 4T1 cells following paclitaxel treatment. Data reflect five biological replicates with surface protein measured as flow cytometric monostains. (F-G) CD69-pos MDA-MB-231 cells express higher levels of Ki67 in untreated and paclitaxel-treated culture conditions. Data reflect six biological replicates. (H-I) CD69-pos 4T1 cells express higher levels of Ki67 in untreated and paclitaxel-treated culture conditions. Data reflect five or four biological replicates, respectively. * p < 0.05; ** p < 0.01; *** p <0.001; **** p < 0.0001. Two-way ANOVA with Dunnett correction (B) or Šidák correction [(C) and (E)], or Student’s t-test [(G) and (I)] Data are means ± SEM.

Figure 5.. MDA-MB-231 cells overexpressing CD69 exhibit increased growth at low density in vitro and in mice.

(A) CRISPR-activation of CD69 via doxycycline exposure in MDA-MB-231 increases its (B) RNA and (C) surface protein expression. Data reflect three biological replicates. (D-E) CD69-activated MDA-MB-231 cells form larger colonies when plated at low density on soft-agar. Data reflect four biological replicates per condition. Scale bar, 2.0 mm. (F) MDA-MB-231 cells were orthotopically implanted into NSG mice for a tumor growth and metastasis assay. Mice were subsequently fed a doxycycline diet on day-8 to induce CD69 in implanted cells. (G-H) CD69-activated MDA-MB-231 cells demonstrated enhanced early tumor growth that (I-J) abated by the study endpoint. Five mice per group. (K) At endpoint, NSG mice implanted with CD69-activated MDA-MB-231 cells had lung metastases that (L) were larger but (M) not more numerous. Individual lesions depicted in (L). Five mice per group. Scale bar, 50 μm. ns = not significant, * p < 0.05; ** p < 0.01; *** p <0.001; **** p < 0.0001. One-way ANOVA with Tukey correction [(B) and (C)] or Dunnett correction (L), Student’s t-test [(E), (H) and (J)], two-way ANOVA [(G) and (I)], or Mann-Whitney test (M). Data are means ± SEM.

Figure 6.. MDA-MB-231 and 4T1 cells magnetically sorted for CD69 grow more efficiently at low density in vitro and in mice.

(A) MDA-MB-231 and 4T1 cells were magnetically sorted into CD69-low and CD69-high fractions. Sorting 200 million cells from each cell line yielded, on average, ~50,000 CD69-high cells that were subjected to enrichment validation via qRT-PCR. MDA-MB-231 sorted populations were used for in vitro growth assays whereas 4T1 populations were assessed for tumor growth at low density in syngeneic BALB/c mice. MDA-MB-231 cells were enriched for (B) CD69 and (C) NFKB1 RNA expression. Data reflect eight biological replicates. (D) Subjecting these CD69-sorted MDA-MB-231 subpopulations to an in vitro growth assay revealed enhanced density by the CD69-high fraction when plated sparsely. Data reflect four biological replicates. (E) The 4T1 CD69-high fraction was enriched for CD69 and (F) NFKB1 RNA expression. Data reflect seven biological replicates. (G) CD69-high 4T1 cells more efficiently form tumors when injected into BALB/c mice at low density with (H) tumors reaching a larger size at endpoint. * p < 0.05; ** p < 0.01; *** p <0.001; **** p < 0.0001. 10 mice per group. Student’s t-test [(B), (C), (E), (F) and (H)], or two-way ANOVA (G) with Šidák correction (D). Data are means ± SEM.

Figure 7.. Knockout of CD69 in MMTV-PyMT-chOVA mice attenuates early tumor growth.

(A) A cell line derived from an MMTV-PyMT-chOVA tumor contains a CD69-pos subpopulation that expands upon paclitaxel treatment and (B) upregulates Ki67. Data reflect six or five biological replicates, respectively. (C) MMTV-PyMT-chOVA mice were bred with CD69-homozygous null (ko/ko) mice to generate MMTV-PyMT-chOVA-CD69^KO/KO mice. (D-E) Comparing spontaneous tumor growth in PyMT-chOVA-CD69^KO/+ versus MMTV-PyMT-chOVA-CD69^KO/KO mice uncovers (F) increased tumor doubling time and (G) slower rate in the latter. PyMT-chOVA-CD69^KO/+, n = 88 tumors in 20 mice. PyMT-chOVA-CD69^KO/KO, n = 119 tumors in 23 mice. Individual tumors depicted in (F) and (G). (H) MMTV-PyMT-chOVA-CD69^KO/KO mice take longer to achieve a tumor burden of 1000 mm³ when compared to mice harboring intact CD69. (I) Tumor cells were dissociated from MMTV-PyMT-chOVA-CD69^KO/+ and MMTV-PyMT-chOVA-CD69^KO/KO mice for orthotopic reinjection back into standard CD69^KO/+ mice. (J) MMTV-PyMT-chOVA-CD69^KO/+ tumor cells are better able to grow upon reinjection into CD69^KO/+ mice and (K) attain larger tumors by study endpoint. PyMT-chOVA-CD69^KO/+ reinjection, n = 14 mice. PyMT-chOVA-CD69^KO/KO reinjection, n = 12 mice. * p < 0.05; ** p < 0.01; *** p <0.001; **** p < 0.0001. Student’s t-test [(A), (B) and (K)], Mann-Whitney test [(F) and (G)], or Mantel-Cox log-rank test (H). Data are means ± SEM.