Targeting metastasis stem cells through a fatty acid receptor (CD36)

#This application is a National Stage Application of PCT/EP2016/073208, filed 29 Sep. 2016, which claims benefit of European Patent Application No. 15382474.3, filed 29 Sep. 2015, which applications are incorporated herein by reference. To the extent appropriate, a claim of priority is made to each of the above-disclosed applications.

#The invention relates to the field of searching for and producing candidates and agents to treat cancer and the control of said disease. More specifically, the invention relates to the use of antibodies and other inhibitors of CD36 activity or expression for the treatment of oral squamous cell carcinoma (OSCC) as well as to the identification of candidates for treatments effective against said cancer and its metastases.

#Squamous cells are a kind of epithelial cell that line several organs of the body, among others: the epidermis, lips and oral cavity. Squamous cell carcinoma (SCC) is a general term for cancers in such cells. Although the terminology is the same, the SCC of different body sites is usually very different with regard to symptoms, prognosis and response to treatment. This is described in detail on the web site of the National Institute of Health (www.cancer.gov/types/head-and-neck/patient/lip-mouth-treatment-pdq) or the professional version of the Merck Manual (www.merckmanuals.com/professional/ear,-nose,-and-throat-disorders/tumors-of-the-head-and-neck/oral-squamous-cell-carcinoma), oral squamous cell carcinoma (OSCC) is a type of head and neck cancer that starts in squamous cells of lips and oral cavity and is the most common cancer of the lips and oral cavity. Smoking and drinking alcohol are important risk factors.

#FIG. 1. OSCCs contain a population of CD44bright label-retaining cells that express a transcriptome signature predominantly associated to lymphatic metastasis and lipid metabolism. A, Detection of LR-CSCs by flow cytometry in SCC-25 xenografts analysed between 4-6 weeks after SCC inoculation (n=6 animals). Numbers indicate CD44brightdye+, CD44brightdye− and CD44dim cells in the represented gate expressed as percentages of total GFP+Lin− SCCs from the parental tumour. (n=3 independent experiments). B, Percentage of dividing cells in the GFP+CD44+DID+ and GFP+CD44+DID− populations analysed by BrdU incorporation and flow cytometry. C, Representation of the quantification of dividing cells in the different populations identified in B: DID+ versus DID−, P<0.05; CD44+ versus CD44−, P<0.05, two-tailed t-test (n=6 independent experiments, 3 mice per experiment). D, Global quantification of CD44brightDye+, CD44brightDye− and CD44dim cells from SCC-pLucGFP xenografts detected with a FACS strategy where viable single cells were selected on the basis of being GFP+ and negative for lineage (Lin) cocktail of antibodies (H2KD, CD31 and CD45) to exclude mouse cells; GFP+Lin− cells were gated for CD44 and dye. E, Representative flow cytometry analysis of the SCC-25 cell line represented in D; percentages from the total GFP+Lin− SCC parental tumour are shown.

#FIG. 2. RT-qPCR validation by human specific TaqMan gene expression assays of differentially expressed genes in the CD44+DID+ and CD44+DID− populations. Data are given as relative expression levels. n=5, P*<0.05, P**<0.005, two-tailed t-test.

#FIG. 3. OSCC LRCs express high levels of CD36, and modulating CD36 expression strongly affects the penetrance and growth of lymph node metastases. A, Detection of CD36+CD44bright cells by flow cytometry of SCC-25, VDH-00 and FaDu xenografts analysed at 4-6 weeks (n=8 animals per cell line). Numbers indicate CD44brightCD36bright, CD44brightCD36low cells in the represented gate, expressed as percentages from the total GFP+Lin− SCC parental tumour. Histograms show the correlation between CD36 expression and DID content for each xenograft. B, Quantification of the correlation between CD36 expression and DID content for SCC-25, JHU-029, Detroit-562, FaDu, VDH-00, VDH-01 and VDH-02 xenografts (n=8 animals per cell line). Numbers indicate percentages from the total GFP+Lin− SCC parental tumour. Results are representative of 3 independent experiments (n=3), P=0.0008, P=0.03, two-tailed t-test. C, D, Bioluminescent monitoring of PMSCV-EV (empty vector) and CD36-OE (over-expression) tumours of SCC-25 (D) (PMSCV-EV, n=7; CD36-OE, n=17) and VDH-00 (D) (PMSCV-EV, n=7; CD36-OE, n=8) xenografts, P<0.0001, two-tailed t-test. Lower graphs show the frequency of developed metastasis in mice expressed as percentages, P=0.0003; P=0.04 two-tailed Fisher exact test. E, Representative haematoxylin and eosin (H&E) staining of lymph nodes from SCC-25 PMSCV-EV and CD36-OE xenografts in C. F, Representation of the CD36 RNA expression level found in cells of xenografts generated from SCC-25 overexpressing CD36, relative to the level found in cells from SCC-25 parental tumours. G, Frequency of developed primary tumours (left chart) or developed metastasis (right chart) from VDH-00 transformed with the PMSCV-EV empty vector (bars labelled “EV”) or with the CD36-OE vector (bars labelled “CD36-OE”. H, Bar charts representing the normalized photon flux observed in primary tumours (left chart) or metastasis (right chart) from VDH-00 transformed with the CD36-OE vector (bars labelled “CD36-OE”), relative to the value observed when the VDH-00 cells have been transformed with the PMSCV-EV empty vector (bars labelled “EV”). I, Bioluminiscent imaging (BLI) and quantification (normalized photon flux) of JHU-029 PMSCV-EV (empty vector) and CD36-OE xenografts, P=0.002; P=0.001; <0.001; <0.0001, two-tailed t-test. J, Representative haematoxylin and eosin (H&E) staining of primary tumours and metastasis from JHU-029 PMSCV-EV and CD36-OE xenografts. The inset shows a magnification of the SCC invading entirely the lymph node in the CD36-OE xenografts (n=5 animals per group). K, Representation of the CD36 RNA expression level found in cells of xenografts generated from SCC-25 overexpressing CD36, relative to the level found in cells from SCC-25 parental tumours. L, Bioluminescent imaging (BLI) of SCC-25 (left column) and JHU-029 (right column) PMSCV-EV (empty vector) and CD36-OE (overexpression) xenografts, M, Frequency of developed primary tumours (left chart) or developed metastasis (right chart) from SCC-25 or JHU-029 (as indicated over the bars) transformed with the PMSCV-EV empty vector (bars labelled “EV”) or with the CD36-OE vector (bars labelled “CD36-OE”). N, Bar charts representing the normalized photon flux observed in primary tumours (left chart) or metastasis (right chart) from SCC-25 or JHU-029 (as indicated over the bars) transformed with the PMSCV-EV empty vector (bars labelled “EV”) or with the CD36-OE vector (bars labelled “CD36-OE”) relative to the value observed when the SCC-25 cells have been transformed with the PMSCV-EV empty vector. PT, primary tumour; LN-Met, lymph node metastasis; Lung-Met, lung metastasis.

#The present invention is based on the findings achieved by the present inventors by using an orthotopic model of OSCC to study the cell cycle heterogeneity of tumour cells in vivo, as described by Myers et al. (2002). With this model, the present inventors have identified a small population of CD36+ cells that contribute to the initiation and growth of OSCC tumours but that are highly predisposed to promote lymph node metastasis.

#Assays described in the Examples of the present application show the effect of modulating the expression/activity of CD36 on primary tumour growth, which is included and quantified for several patient-derived tumour and established cell lines. It can be seen that some primary oral tumours respond better than others to CD36 inhibition, but the overall conclusion found by the present inventors is that depletion of endogenous CD36 in vivo by shRNA or blocking of activity by means of neutralizing antibodies slightly reduces primary tumour burden.

#However, the present inventors have found that, in every single tumour tested, inhibition of CD36 (both by antibodies neutralizing its activity or by shRNAs) has a dramatic effect regarding metastasis initiation and progression, decreasing metastatic penetrance and growth of all cell lines and patient-derived tumours that they have tested. This is a very surprising effect because, as it has been discussed above, an inverse correlation had been previously reported for CD36 expression with regard to metastatic potential of different tumours and low CD36 gene expression has been found to correlate with a higher metastasis grade in other metastatic cancer such as colon and ovarian cancers and its expression is almost absent in highly aggressive breast cancer lines.