PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer.

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRβ-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC

Combinations of Toll-like receptor 8 agonist TL8-506 activate human tumor-derived dendritic cells

BackgroundDendritic cells (DCs) are professional antigen presenting cells that initiate immune defense to pathogens and tumor cells. Human tumors contain only few DCs that mostly display a non-activated phenotype. Hence, activation of tumor-associated DCs may improve efficacy of cancer immunotherapies. Toll-like receptor (TLR) agonists and interferons are known to promote DC maturation. However, it is unclear if DCs in human tumors respond to activation signals and which stimuli induce the optimal activation of human tumor DCs.MethodsWe first screened combinations of TLR agonists, a STING agonist and interferons (IFNs) for their ability to activate human conventional DCs (cDCs). Two combinations: TL8-506 (a TLR8 agonist)+IFN-γ and TL8-506+Poly(I:C) (a TLR3 agonist) were studied in more detail. cDC1s and cDC2s derived from cord blood stem cells, blood or patient tumor samples were stimulated with either TL8-506+IFN-γ or TL8-506+Poly(I:C). Different activation markers were analyzed by ELISA, flow cytometry, NanoString nCounter Technology or single-cell RNA-sequencing. T cell activation and migration assays were performed to assess functional consequences of cDC activation.ResultsWe show that TL8-506 synergized with IFN-γ or Poly(I:C) to induce high expression of different chemokines and cytokines including interleukin (IL)-12p70 in human cord blood and blood cDC subsets in a combination-specific manner. Importantly, both combinations induced the activation of cDC subsets in patient tumor samples ex vivo. The expression of immunostimulatory genes important for anticancer responses including CD40, IFNB1, IFNL1, IL12A and IL12B were upregulated on stimulation. Furthermore, chemokines associated with CD8$^{+}$ T cell recruitment were induced in tumor-derived cDCs in response to TL8-506 combinations. In vitro activation and migration assays confirmed that stimulated cDCs induce T cell activation and migration.ConclusionsOur data suggest that cord blood-derived and blood-derived cDCs are a good surrogate to study treatment responses in human tumor cDCs. While most cDCs in human tumors display a non-activated phenotype, TL8-506 combinations drive human tumor cDCs towards an immunostimulatory phenotype associated with Th1 responses on stimulation. Hence, TL8-506-based combinations may be promising candidates to initiate or boost antitumor responses in patients with cancer

Hepatic stellate cells suppress NK cell-sustained breast cancer dormancy

The persistence of undetectable disseminated tumour cells (DTCs) after primary tumour resection poses a major challenge to effective cancer treatment; 1-3; . These enduring dormant DTCs are seeds of future metastases, and the mechanisms that switch them from dormancy to outgrowth require definition. Because cancer dormancy provides a unique therapeutic window for preventing metastatic disease, a comprehensive understanding of the distribution, composition and dynamics of reservoirs of dormant DTCs is imperative. Here we show that different tissue-specific microenvironments restrain or allow the progression of breast cancer in the liver-a frequent site of metastasis; 4; that is often associated with a poor prognosis; 5; . Using mouse models, we show that there is a selective increase in natural killer (NK) cells in the dormant milieu. Adjuvant interleukin-15-based immunotherapy ensures an abundant pool of NK cells that sustains dormancy through interferon-γ signalling, thereby preventing hepatic metastases and prolonging survival. Exit from dormancy follows a marked contraction of the NK cell compartment and the concurrent accumulation of activated hepatic stellate cells (aHSCs). Our proteomics studies on liver co-cultures implicate the aHSC-secreted chemokine CXCL12 in the induction of NK cell quiescence through its cognate receptor CXCR4. CXCL12 expression and aHSC abundance are closely correlated in patients with liver metastases. Our data identify the interplay between NK cells and aHSCs as a master switch of cancer dormancy, and suggest that therapies aimed at normalizing the NK cell pool might succeed in preventing metastatic outgrowth

Hormone-sensing cells require Wip1 for paracrine stimulation in normal and premalignant mammary epithelium

10.1186/bcr3381Breast Cancer Research15

Expression analysis of rare cellular subsets: Direct RT-PCR on limited cell numbers obtained by FACS or soft agar assays

10.2144/000114019BioTechniques544208-211BTNQ

Transcriptional Repressor Tbx3 Is Required for the Hormone-Sensing Cell Lineage in Mammary Epithelium

<div><p>The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development) and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production). Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER) and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells.</p></div

Tbx3 is required for the generation of hormone-sensing cells.

<p>(A) Primary MECs from wildtype or KI mice were transduced with a non-silencing lentiviral vector (control) or with two independent short hairpins against Tbx3 (sh-1 and sh-2). Cells were injected into mammary fat pads devoid of endogenous epithelium and outgrowths were analyzed 8–10 weeks later for the identity of lentivirally transduced cells (recognized by tGFP expression). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110191#pone.0110191.s004" target="_blank">File S4B</a> for a schematic experimental design. Each bar represents one fat pad and 46 to 569 tGFP+ luminal cells were counted per fat pad. There is a significant bias against the formation of HS cells by cells with Tbx3 knockdown (Chi square of shRNA versus control transplant <0.01). (B) Paraffin section of a cleared mammary fat pad transplanted with MECs that were exposed to the non-silencing control vector. Transduced cells are identified with an antibody staining against tGFP (green), luminal cells are identified by cytokeratin 8 (blue) and HS cells are identified by the estrogen receptor (ER, red). White arrow indicates transduced cells contributing to the hormone-sensing lineage. (C) Paraffin section of a mammary fat pad transplanted with MECs exposed to the first short hairpin against Tbx3. White arrow head indicated transduced cells in the luminal alveolar (ER-negative) lineage. The background of immunohistochemistry is higher in transplanted samples (arguably due to fibrosis). Where ER staining was ambiguous due to high background, we used progesterone receptor (PR, red) staining as an alternative marker for HS cells. (D) Paraffin section of a mammary fat pad transplanted with MECs exposed to the second short hairpin against Tbx3. White arrow head indicates transduced cells in the luminal alveolar (ER-negative) lineage. White scale bar is 20 µm and yellow scale bar is 10 µm.</p

Fluorescent reporter reveals distinct Tbx3 expression in mammary epithelial cell subsets.

<p>(A) Epithelial cells isolated from mammary glands of wildtype (Tbx3^+/+) or knock-in (Tbx3^+/Venus) mice show three peaks with different levels of Venus expression (Low, Medium and High). (B) Mammary epithelial cells (MECs) from 3 independent Tbx3^+/Venus animals were sorted according to Venus signal intensity. qPCR on 500 directly lysed cells shows that both Venus and Tbx3 mRNA correlates tightly with Venus fluorescence intensity. (C) MECs were labeled with fluorescent antibodies against CD24 and α6-integrin (CD49f) to distinguish the luminal (blue) and basal (red) cell populations. (D) When plotted separately, luminal cells from Tbx3^+/Venus mammary glands show two main populations; Venus^Low (cells with low Tbx3 expression) and Venus^High (cells with high Tbx3 expression). Basal cells express intermediate level of Venus (and Tbx3). (E) Quantification of the percentage of Venus-Low, -Medium and -High cells in the luminal and in the basal population of Tbx3^+/Venus epithelium. Data are presented as mean ± SD of three individual adult virgin Tbx3^+/Venus animals. (F) Populations gated based on Tbx3 expression (Venus-Low, -Medium and -High) plotted on a CD24/CD49f contour plot.</p

Tbx3 expression in epithelial cells in intact mammary glands.

<p>(A–C) Confocal immunofluorescence on paraffin sections of mammary glands from wildtype adult virgin mice. (A) Ductal structure probed with antibodies for Tbx3 (green), the estrogen receptor (ER, red) and the luminal cell marker cytokeratin-8 (CK8, blue). Nuclei are stained with DAPI (grey). (B) Duct probed for Tbx3 (green), the basal marker smooth muscle actin (SMA, red) and CK8 (blue). (C) Duct probed for Tbx3 (green), E-Cadherin (E-cad, red) and CK8 (blue). Images are representative of staining performed on paraffin sections of 3 independent animals. Scale bar is 20 µm or 10 µm for the inset.</p

Tbx3 marks the hormone sensing lineage, including ER+ progenitor cells.

<p>(A) Combined density/contour plot of mammary epithelial cells (from a pool of 5 Tbx3^+/Venus mice) separated into basal (red) and luminal (blue) cells based on CD24 and alpha6-integrin (CD49f) expression. (B) Colony forming potential of 1000 sorted cells from each population, representative of two independent experiments. (C) Histogram of luminal mammary epithelial cells Venus^High (luminal) and Venus^Low (luminal) cells sorted for colony forming assay (from a pool of 5 Tbx3^+/Venus mice per experiment). (D) Colony forming potential of 1000 sorted cells from each population, representative of two independent experiments. (E) FACS profile of hormone-sensing (CD49b^high and CD49b^low) and alveolar progenitor cells that were used for colony assays. Cells are color-coded based on Venus expression (green = Venus^High, grey = Venus^Low). (F) Fold change in Tbx3, progesterone receptor (PR) and estrogen receptor (ER) mRNA expression of sorted CD49b^high and CD49b^low hormone-sensing cells and alveolar progenitor cells, relative to CD49b^low hormone-sensing cells (dark purple bar). Fold change in Elf5 and cKit mRNA expression is shown relative to luminal alveolar cells (orange bar). (G) Colony forming potential of 1000 sorted luminal cells: CD49b^low and CD49b^high hormone-sensing cells and alveolar progenitor cells. (H) Quantification of colony forming assays with HS cells (Sca1^highCD49b^low), HS progenitor cells (Sca1^highCd49b^high) and alveolar progenitor cells (Sca1^lowCD49b^high). Bars represent the mean of three independent pools of 5–6 adult virgin Tbx3^+/Venus animals ± SD. HS progenitor cells form more colonies than HS cells (p = 0.02, paired t-test) and there is no significant difference in colony forming potential between HS progenitor and alveolar progenitor cells (p = 0.21, paired t-test).</p