Tumor-associated macrophages infiltrate plasmacytomas and can serve as cell carriers for oncolytic measles virotherapy of disseminated myeloma

Abstract

In multiple myeloma, some of the neoplastic plasma cells are diffusely dispersed among the normal bone marrow cells (bone marrow resident), whereas others are located in discrete, well-vascularized solid tumors (plasmacytomas) that may originate in bone or soft tissue. Interactions between bone marrow-resident myeloma cells and bone marrow stromal cells (BMSCs) are important determinants of myeloma pathogenesis. However, little is known of the factors sustaining myeloma growth and cell viability at the centers of expanding plasmacytomas, where there are no BMSCs. Histologic sections of 22 plasmacytomas from myeloma patients were examined after immunostaining. Abundant CD68+, CD163+, S100-negative macrophage infiltrates were identified in all tumors, accompanied by scattered collections of CD3+ T lymphocytes. The CD68+ tumor-associated macrophages (TAM) accounted for 2-12% of nucleated cells and were evenly distributed through the parenchyma. The TAM generally had dendritic morphology, and each dendrite was in close contact with multiple plasma cells. In some cases, the TAM were strikingly clustered around CD34+ blood vessels. To determine whether cells of the monocytic lineage might be exploitable as carriers for delivery of therapeutic agents to plasmacytomas, primary human CD14+ cells were infected with oncolytic measles virus and administered intravenously to mice bearing KAS6/1 human myeloma xenografts. The cell carriers localized to KAS6/1 tumors, where they transferred MV infection to myeloma cells and prolonged the survival of mice bearing disseminated human myeloma disease. Thus, TAM are a universal stromal component of the plasmacytomas of myeloma patients and may offer a promising new target for therapeutic exploitation. Am. J. Hematol. 2009. (c) 2009 Wiley-Liss, Inc.

Figure 1

Leukocyte infiltrates in plasmacytomas from multiple myeloma patients. (A) Hematoxylin and eosin stained, (B–I) CD68 immunostained or (J–L) CD3 immunostained paraffin sections of plasmacytomas from representative cases. All immunostained slides were counterstained with hematoxylin. Note that, in general, the CD68 cells are uniformly dispersed within the plasmacytoma, whereas some of the CD3 cells are found in clusters. Original magnification is 200×.

Figure 2

Quantitation of CD68+ infiltrates in plasmacytomas. (A) Representative high-power fields (HPF, 400× magnification) showing images from two different plasmacytoma cases immunostained with anti-CD68. Note the dendritic morphology of the macrophages. (B) Percentage of CD68-positive cells in the 22 cases of plasmacytomas examined. Numbers of CD68-positive cells in 10 representative HPFs were counted for each case and expressed as a percentage of the total number of nucleated cells in the same field. Error bars represent the SD about the mean (n = 10 HPFs).

Figure 3

Spatial relationship between tumor-associated macrophages (TAM) and tumor blood vessels. Plasmacytomas were dual stained with anti-CD68 (Fast red, red staining) and anti-CD34 (DAB, brown staining) antibodies to detect TAMs and tumor blood vessels, respectively. (A and B) Representative examples showing independent distributions of CD68 macrophages and tumor blood vessels. Here, the TAM are not associated with the blood vessels. This pattern was observed in the majority of the cases. (C and D) Examples in which the TAM are closely associated with the CD34 blood vessels with evidence of vascular mimicry. (D and F) Two patterns of TAM distribution are seen in these examples. Here, one population of TAM is distributed randomly and another population is closely associated with the blood vessels. Original magnification is 200×.

Figure 4

Potential use of monocytes and dendritic cells (DC) as virus-infected cell carriers. (A) Susceptibility of CD14+ monocytes, iDCs, and mDCs from four blood donors to infection by MV-eGFP. Percentage of infected GFP-positive cells was analyzed by flow cytometry at 48 hr postinfection. (B) Heterofusion of MV-Luc–infected DiO (green)-labeled iDCs with RFP-expressing (red) human KAS 6/1 myeloma cells in culture, resulting in syncytium formation at 24 and 48 hr post– cell mixing. Mock infected iDC were used as controls (lower panels). (C) Serial SPECT-CT images of a mouse that has received an intravenous dose of Indium-111 labeled iDC from 30 min to Day 3 post–cell delivery. (D) Scintillation counts (counts per minute) in the respective organs of mice that received an intravenous dose of lndium-111 labeled CD14, iDC, or mDC at 24 hr post–cell delivery. The amount of radioactivity in the respective organs was calculated as a percentage of the radioactivity present in the whole animal at 24 hr.

Figure 5

Adoptively transferred iDC delivered MV to myeloma tumors and extended survival of mice with systemic myeloma disease. (A) Serial bioluminescent imaging of a SCID mouse given one intravenous dose of 1 × 10⁶ MV-Luc–infected iDCs at various time intervals. The positions of the lungs and subcutaneous KAS 6/1 tumor xenograft are noted. (B) Confocal microscope images of tumors harvested from mice that received uninfected or MV-infected iDCs at 6 hr postadoptive transfer of iDCs. The tissue sections were stained with anti-MHC II antibody (red staining) to detect human iDCs. Magnification is 200×. (C) Gross examination of tumors from two different mice (M#5, M#6) harvested 3 days postinfusion of MV-RFP–infected iDCs revealed the presence of single RFP-positive infected iDCs and RFP-positive syncytia. Freshly harvested tumors were cut into 5-mm slices and photos were taken using a Nikon fluorescence microscope. Magnification is 100×. Immunohistochemical staining for MV-N protein (green, Alexa-488) in 5-µm cryosections confirmed the presence of MV infection in the tumor sections. Note the green infectious foci of MV infection. Magnification is 200×. (D) Kaplan–Meier survival curve of SCID mice bearing systemic KAS 6/1 tumors. Mice received two doses of saline (n = 20 mice) or MV-NIS–infected iDC (n = 15 mice), given 1 week apart.