POS – TEST

One of the main research aims of this thesis was to establish effective loss-of-function experimental systems allowing for the specific dissection of stromal CAV1 contribution to tumor progression in vivo. In order to tackle these requirements, two different complementary approaches were devised.

3.1.1 Engineering promoter-based fibroblast selectivity in dual-reporter miRNA-based lentiviral vectors

In a first model, breast cancer biopsy fragments would have been orthotopically transplanted into immunodeficient NSG mice and transduced intratumorally with lentiviral vectors targeting the CAV1 mRNA, selectively in fibroblasts (Fig. 11A). In this strategy, human breast tumor biopsies, containing both patient stroma and cancer cells, would have been engrafted onto immunodeficient NSG mice as a means to expand the small biopsies into tumor masses. Subsequently, the resulting tumors would have been subdivided and frozen to set up a small tumor biobank that would have later been used to transplant and perform in vivo intratumoral lentiviral injections.

In order to selectively target stromal fibroblasts, present in the tumor biopsy, a lentiviral vector expressing a CAV1-targeting microRNA-like (miRNA) under the control of a fibroblast-specific promoter, was developed (Fig. 11B and Table S4). This lentiviral vector also contained a bicistronic cassette, linked by a self-cleaving P2A peptide357_{, composed of a CherryFP} fluorescent protein, for in vitro studies, and a far-red light emitting luciferase (RE8luc)358 optimized for in vivo tracking. Downstream of the expression cassette, either a non-targeting scramble miRNA or four different human CAV1 targeting miRNAs were cloned.

The inclusion of the CAV1 targeting sequences into a miRNA frame, as opposed to RNApol III-based silencing systems (shRNA), allows for the simultaneous transcription of the expression cassette (CherryFP - P2A - RE8luc, aka ChFP2A-RE8luc) and the silencing

element (miRNA) under the control of a unique upstream RNApol II-driven promoter360_{. Thus,}

the detection of in vitro fluorescence or in vivo bioluminescence would be indicative of ongoing miRNA transcription.

A first vector version was constructed to bear a constitutive cytomegalovirus promoter (PCMV) to check the downregulation efficiency of all four different miRNA sequences targeting CAV1. The miR30.CAV1.2 sequence showed the highest silencing efficiency as compared with the

63 Standard lentiviral vector packaging systems do not confer cell type-level selectivity for infection. Thus, adapting a fibroblast-specific promoter that would actively drive the expression of the integrated provirus selectively in stromal fibroblasts was paramount. In order to screen for fibroblast-specific transcripts and promoters, the expression of a number of published fibroblast-specific markers (FAP, FSP1, and ITGA11) was assessed both in cancer-associated and normal fibroblasts and in different breast cancer cell lines (Fig. 11C).

Figure 11. Engineering promoter-based fibroblast selectivity in dual-reporter miRNA-based lentiviral vectors. (A) In vivo targeting of breast tumor stromal fibroblasts. Depiction of immunodeficient NSG mice implanted with tumors derived from human biopsies (xenografts) and intratumorally injected with lentivirus in which fluorescent protein/luciferase and microRNA expression are controlled by a fibroblast-specific promoter. (B) Third generation lentivirus expressing a bicistronic cassette, containing a: (i) fluorescent protein (CherryFP, in red) and a light-emitting luciferase (RE8luc, in dark red) linked via a P2A self-cleaving peptide (in white), and (ii) a miR30-based shRNA silencing element. Expression of both elements (i and ii) is controlled either by a tissue-specific promoter (PITGA11, lower scheme)

inserted into a multicloning site (MCS, in green, upper scheme) or a ubiquitous PCMV promoter. Unique

restriction enzyme sites are depicted below the schemes and the rest of lentiviral vector elements (LTRs, Ψ, RRE, cPPT, and WPRE) are shown in grey. (C) Expression levels of CAV1, ITGA11, FAP and FSP1 in different CAFs cell lines, normal (NFs) skin fibroblasts (SkinFib) and breast cancer cell lines (brCAN).

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Heatmap shows high (in red) and low (in blue) expression levels of mRNA expression assayed by qPCR (ITGA11 was assayed using 5 different primers, PB2-6). Western blot depicts protein levels of ITGA11 in the aforementioned cells; GAPDH was used as loading control. (D) Representative fluorescence microscopy images of brCAN (MCF7) and CAFs (1068sk) co-cultures infected with a CherryFP (in red) containing lentivirus (ChFP2A-RE8luc) expressed under the control of PCMV (left panel) or ITGA11

promoter (right panel). Pan-cytokeratin, an epithelial marker of brCAN is shown in green. Scale bar: 20µm. (E) Western blot showing CAV1 expression levels in HeLa and PC-3 cells after infection with either non-targeting (miR30.scr) or CAV1-targeting (miR30.CAV1) PCMV ChFP2A-RE8luc lentivirus.

While Fibroblast Specific Protein 1 (FSP1) is a widely used fibroblast marker361_{, not all isolated} fibroblasts exhibited significant expression. Moreover, “mesenchymal-like” breast cancer cell

lines such as MDA-MB-231 did express detectable levels (Fig. 11C). Finally, ITGA11 promoter

was chosen over FAP promoter given the overall higher expression of its mRNA in CAFs (Fig.

11C).

The specificity of ChFP2A-RE8luc lentivirus containing the ITGA11 promoter was tested in an

in vitro co-culture system of MCF7 breast cancer and CCD1068SK CAF cell lines, showing a

lack of CherryFP expression in cancer cells and CherryFP production in fibroblasts (Fig.11D).

Figure 12. CAFs – CAV1 targeting in CAFs-brCAN co-culture assay. (A) Fluorescence microscopy tilescan reconstruction of brCAN/CAFs co-culture assays. CCD 1068SK CAFs show expression of CAV1 (in green) while MCF7 brCAN cells do not. MCF7 are marked with an antibody against Pan- Cytokeratin, an epithelial cell marker (in magenta). Infected cells are expressing CherryFP (in red) and nuclei are stained with DAPI (in blue). (B) Representative images of brCAN/CAFs co-culture assays. CAV1 levels in cells infected with non-targeting CMV miR30.scr (left panel, white arrow marked, in red) remain unaffected. CAFs expressing CAV1 targeting miRNAs under the control of a PCMV promoter

(central panel, white arrow, in red) downregulate CAV1 levels. Co-cultures infected with ITGA11 promoter controlled lentivirus (right panel, in red), while maintaining CAF expression specificity, showed poor CAV1 silencing efficiency.

65 As the objective of this lentiviral system was to achieve CAV1 silencing specifically in CAFs, while leaving brCAN cells unaffected, miRNA-expressing ChFP-P2A-RE8luc lentiviral vectors, either under the control of the ubiquitous PCMV or the fibroblast-specific ITGA11 promoter were tested in the same CAF/brCAN co-culture scenario (Fig. 12A). CAFs proved difficult to transduce, as seen in the infections with the PCMV version of the lentivirus, and while ITGA11 remained a good promoter choice for fibroblast-specific expression, CAV1 silencing efficiency was unsatisfactory in these systems as most infected CAFs (CherryFP positive) displayed

detectable residual CAV1 expression (Fig. 12B).

Given the technical challenges posed by the first approach, a second strategy that overcame the hurdles of a potential suboptimal in vivo CAF infection and CAV1 downregulation was finally chosen.

3.1.2 Efficient CAV1 silencing ex vivo (pre-grafting) in CAFs for comparative xenograft studies

In this second strategy, primary breast stromal CAFs and TNBC breast cancer cell lines (TCs) were modified ex vivo prior to their injection into the mammary fat pad of NSG mice (Fig. 13A). Two stable CAF cell lines derived from human primary breast CAFs expressing high levels of CAV1 were generated: (i) CAFscr, infected with a non-targeting lentivirus, and (ii)

CAFshCAV1, where CAV1 was silenced using a lentiviral construct (Fig. 13C).

Figure 13. Efficient CAV1 silencing ex vivo (pre-grafting) in CAFs for comparative xenograft studies. (A) Experimental approach to generate mouse xenograft models of human breast cancer with high and low stromal levels of CAV1. Three different cell combinations were injected into the mammary fat pads of NSG mice: (i) isolated luciferase-labeled breast cancer cells (TCsluc_{) without a stromal}

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component (CAFs), (ii) TCsluc _{with CAFscr, constituting the high stromal CAV1 xenografts (sCAV1high),}

and (iii) TCsluc _{with CAFshCAV1, as low stromal CAV1 tumors (sCAV1low). (B) Western blot showing}

CAV1, ITGA11 and ACTA2 expression levels in brCAFs cells after infection with either non-targeting (miR30.scr) or CAV1-targeting (miR30.CAV1.2-4) PCMV ChFP2A-PuroR lentivirus. ITGA11 (Integrin α11)

and ACTA2 (Smooth muscle actin) are considered normal- and cancer-associated fibroblast markers respectively. (C) Lentiviral vector pLVX-CMV-ChFP2A-RE8luc, aimed at miRNA expression (non- targeting or CAV1 targeting) from Fig. 11B where the luciferase gene has been swapped with a puromycin resistance gene (PuroR_{) allowing antibiotic-mediated selection of transduced cells. (D)}

Lentiviral vector used to generate fluorescent protein/luciferase-labeled TCs (TCsluc_{). Its bicistronic}

expression cassette contains a: (i) fluorescent protein (CherryFP, in red), for FACS sorting, and a luciferase (RE8luc, in dark red) for in vivo tracking, linked via a P2A self-cleaving peptide (in white); all under the control of an ubiquituos PCMV promoter.

Then, three different combinations of human cells were injected into the mammary fat pad of immunodeficient NSG mice: (i) either luciferase-labeled human breast cancer cell lines (TCsluc_: MDA-MB-436 or MDA-MB-468), (ii) breast cancer cells (TCs) plus CAFscr fibroblasts or (iii) breast cancer cells (TCs) together with CAFshCAV1 fibroblasts. Tumor xenografts containing CAFscr or CAFshCAV1 fibroblasts were considered as high (sCAV1high) or low stromal CAV1

(sCAV1low) tumors respectively (Fig. 13A).

For the generation of CAFscr and CAFshCAV1 cells, a modified version of the lentivirus designed in the first approach was used (Fig. 11B). In this lentivirus the luciferase cassette was substituted by a puromycin resistance gene (PuroR_{) allowing an antibiotic-mediated} selection of infected cells. In this case, as expression specificity was no longer needed (infection was performed in vitro in non-mixed cell populations), both the expression cassette

(ChFP2A-PuroR_{) and the miRNA-based silencing elements were under the control of an}

ubiquitous PCMV promoter (Fig. 13C).

Lentivirus-mediated CAV1 silencing in CAFs was performed using 3 different miRNA sequences (miR30.CAV1.2-4), again showing the highest CAV1 silencing levels in the case of

miR30.CAV1.2 (Fig. 13B). Primary human breast CAFs infected with miR30.scr were labeled

CAFscr, while the ones infected with miR30.CAV1.2 were used for the rest of the study as CAFshCAV1 given they had the highest level of CAV1 silencing.

In order to track cancer cell growth in vivo, luciferase-labeled breast cancer cell lines were

generated (TCsluc_{) from established TNBC cell lines (MDA-MB-436 and MDA-MB-468) via}

lentiviral infection. The lentiviral vector used in the infection of TCs (pLVX-CMV-ChFP2A- RE8luc) expressed a CherryFP fluorescent protein, that allowed for FACS sorting of infected cells, and a far-red light emitting luciferase (RE8luc) optimized for in vivo tracking of labeled

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