Fig. 4: Subcutaneously transplanted agnospheres reproduce the early and multi-organ metastatic pattern of CUP patients.

a Outline of longitudinal in vivo monitoring of spheropatients transplanted subcutis with luciferase-labeled agnospheres, followed by endpoint ex vivo organ imaging and histopathological analysis. IS-tumor injection site tumor. b Representative in vivo images of mice transplanted with AS901, showing increasing bioluminescent signals at metastatic sites at the indicated time points (d, days). Black tape was applied to mask the IS-tumor residual signal. c Representative macroscopic images of the most frequent metastatic sites. Top: bioluminescent signals of different explanted organs; bottom: photographs of macrometastases. d Histopathology (H&E) and immunohistochemistry for human CK18 or human Vimentin, revealing metastases in the indicated organs of mice transplanted with AS901 or AS906, respectively. Scale bar, 50 μm. Representative experiments are shown (at least n = 3 stainings in independent organs were obtained with similar results). e Bar graph showing the percentage of mice transplanted subcutis with agnospheres or colosphere mCRC729 that developed IS-tumors and/or metastases (AS901: n = 33, AS906: n = 50; AS43: n = 44; N-AS47: n = 13; mCRC729: n = 14). f Bar graph showing the percentage of mice subcutaneously (s.c.) transplanted with agnospheres as in e that displayed metastasis (mts) at each of the listed sites. g Immunostainings for pan-cytokeratin AE1/AE3 showing tumor cells disseminated in organs explanted 10 days after AS43 subcutaneous injection. Scale bar, 50 μm (n = 4 independent experiments with similar results were obtained).