Early tumor microenvironment events following RV + BTZ combination therapy in vivo. (A) Schematic representation of treatment time line in mice. (B) To evaluate early tumor microenvironment events, mice were euthanized after 4 days of treatment; their spleens and BM were then formalin fixed and paraffin embedded. Serial sections were stained and scored for various markers (supplemental Table 1) per previously published IHC protocols.^54-56  Optimal conditions were determined using various myeloma cell lines with different sensitivities infected with RV, and included a dilution of 1:1000 and a rabbit/anti-goat secondary antibody (ABCAM, 1:500 dilution). Myeloma tumor burden in the treated cohorts for Figures 2B-F and 3-5 are given in Table 3. Reoviral capsid protein was detected within myeloma cells in scattered foci in mice treated only with LV (arrows), whereas many more viral positive myeloma cells were seen in the spleen after live virus and BTZ treatment (magnification ×200 = ×10 [ocular lens] and ×20 [objective lens]). (C) LV + BTZ combination therapy leads to significantly higher RV protein production. Bar diagrams representing quantified LV RNA and protein in BM and spleen myeloma tumor. N = 3; ***P < .001, Conover’s test. (D) Splenic myeloma caspase 3 expression images of IHC sections. Note the low activated caspase-3 expression in the mice treated with the VC or DV. Foci of myeloma cells positive for caspase-3 were seen in the spleen of the mice treated with LV alone, whereas there was a diffuse and strong increase in caspase-3 expression in the mice treated with LV and BTZ (magnification ×200 = ×10 and ×20). (E) Significantly higher caspase 3 is expressed in MM tumor of LV + BTZ combination therapy. Bar diagrams representing quantified caspase 3. N = 3; ***P < .001, **P < .01, *P < .05, Conover’s test. (F) Coexpression of CD138 and caspase 3 in spleen myeloma cells. Coexpression of caspase 3 in CD138⁺ myeloma cells (right, fluorescent yellow) in RV + BTZ–treated spleens confirms that it is primarily myeloma cells that undergo apoptosis (magnification ×400 = ×10 and ×40). (B,D) Camera: Ventana Vias Zeiss Axio; acquisition software: Nuance. Composite figure via Adobe Photoshop CS4. PFU, plaque-forming unit; TFC, tumor-free control.