Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model

Mesenchymal stromal cells (MSCs) are considered an important therapeutic tool in cancer therapy. They possess intrinsic therapeutic potential and can also be in vitro manipulated and engineered to produce therapeutic molecules that can be delivered to the site of diseases, through their capacity to home pathological tissues. We have recently demonstrated that MSCs, upon in vitro priming with anti-cancer drug, become drug-releasing mesenchymal cells (Dr-MCs) able to strongly inhibit cancer cells growth

Single-Shot Local Injection of Microfragmented Fat Tissue Loaded with Paclitaxel Induces Potent Growth Inhibition of Hepatocellular Carcinoma in Nude Mice

Hepatocellular carcinoma (HCC) is poorly beneficiated by intravenous chemotherapy due to inadequate availability of drugs at the tumor site. We previously demonstrated that human micro-fragmented adipose tissue (MFAT) and its devitalized counterpart (DMFAT) could be effective natural scaffolds to deliver Paclitaxel (PTX) to tumors in both in vitro and in vivo tests, affecting cancer growth relapse. Here we tested the efficacy of DMFAT-PTX in a well-established HCC in nude mice. MFAT-PTX and DMFAT-PTX preparations were tested for anti-cancer activity in 2D and 3D assays using Hep-3B tumor cells. The efficacy of DMFAT-PTX was evaluated after a single-shot subcutaneous injection near a Hep-3B growing tumor by assessing tumor volumes, apoptosis rate, and drug pharmacokinetics in an in vivo model. Potent antiproliferative activity was seen in both in vitro 2D and 3D tests. Mice treated with DMFAT-PTX (10 mg/kg) produced potent Hep-3B growth inhibition with 33% complete tumor regressions. All treated animals experienced tumor ulceration at the site of DMFAT-PTX injection, which healed spontaneously. Lowering the drug concentration (5 mg/kg) prevented the formation of ulcers, maintaining statistically significant efficacy. Histology revealed a higher number of apoptotic cancer cells intratumorally, suggesting prolonged presence of PTX that was confirmed by the pharmacokinetic analysis. DMFAT may be a potent and valid new tool for local chemotherapy of HCC in an advanced stage of progression, also suggesting potential effectiveness in other human primary inoperable cancers

Transforming growth factor-beta1 and CD105 promote the migration of hepatocellular carcinoma-derived endothelium.

Abstract Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming growth factor-beta1 (TGF-beta1) and its coreceptor CD105 have been shown to contribute to HCC malignant progression. TGF-beta1 and CD105 have also been implicated in angiogenesis, but their role in the vascularization of HCC has not been investigated. To fill this gap, we studied the effect of TGF-beta1 and CD105 on HCC-derived endothelium. By using immunomagnetic beads, we isolated and cultured endothelial cells (ECs) from HCC (HCC-EC) and adjacent nonneoplastic tissue (nNL-ECs) obtained from 24 liver biopsies. HCC and nNL biopsies were also analyzed by immunohistochemistry for the expression of CD105, TGF-beta1, Ve-cadherin (Ve-cad), CD44, beta-catenin, and E-cadherin. Compared with nNL-ECs, HCC-ECs had higher expression of CD105, enhanced spontaneous motility, and greater capacity to migrate in response to TGF-beta1 (5 ng/mL), particularly in the presence of a fibronectin matrix. The chemotactic effect of TGF-beta1 was blocked by anti-CD105 antibodies and correlated with the grade of HCC malignancy. Histologic examination of HCC biopsies showed that HCCs with the worse malignant features had the highest expression of TGF-beta1, CD105, and angiogenic markers (Ve-cad and CD44). Because CD105 was highly expressed in microvessels at the tumor periphery and TGF-beta1 staining was only found in neoplastic hepatocytes, we conclude that HCC-derived TGF-beta1 may act as a chemoattractant for CD105-expressing ECs and as a promoter of tumor angiogenesis. Thus, drugs that selectively target the TGF-beta1/CD105 axis may interfere with HCC-related angiogenesis and HCC progression

Additional file 2: Figure S1. of Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model

SR4987GFP and SR4987GFP-PTX arrest in the lung of mice upon i.v. administration. Mice were sacrificed at 6 h, 24 h and 48 h after treatments with cells. Panel (A) shows photos taken under fluorescent microscopy immediately upon enzymatic lung tissue disruption, showing the presence of several fluorescent cells until 48 h after their injection. Not significant difference were noted between mice treated with SR4987GFP and SR4987GFP-PTX (10x magnification). Panel (B) shows photos of SR4987GFP and SR4987GFP-PTX cultured for 2 and 7 days upon isolation from mice, indicating that cells maintain their viability (20x magnification). Figure S2. Sca-1 is highly expressed on SR4987 and can be used for their detection in the mice lung. In (A) FC analysis showing the high positivity of the SR4987 for Sca-1. In (B), Immunostaining for anti-Sca-1 of lungs derived from mice treated i.v. with saline , 106 SR4987 and 106 SR4987PTX cells and sacrificed 24 h after treatments. Pictures show the high presence of Sca-1+ cells (black arrows) in the parenchyma and in the lumen of lung vessels in mice treated with both SR4987 and SR4987PTX. Only, very few Sca-1+ cells were detected in control mice (CTRL) (photo 20x, in the box 40x magnifications). Figure S3. Sensibility of SR4987 to PTX and efficacy of SR4987PTX-CM to inhibit B16 proliferation. In (A) the proliferation of SR4987 in the presence of increasing concentration of PTX. The IC50 of PTX was around 34 ng/ml. In (B) is shown that viability of SR4987 is not affected by PTX even at concentration up 5000 ng/ml. In (C) is shown the growth inhibition of Molt-4 tumor cells in the presence of decreasing dilution of SR4987PTX-CM confirming that SR4987PTX release significant amount of PTX in the CM. In (D) the inhibition of proliferation of B16 cells in the presence of increasing concentration of PTX. The IC50 is 7.18 ± 4.99 ng/ml. In (E) is shown the dose dependent efficacy of SR4987PTX-CM to inhibit B16 cells proliferation with an IC50 DIL of around 1/10. Figure S4. Co-culture of SR4987 and SR4987PTX with B16 cells using transwell insert. B16 cells seeded on the top of membrane (0.4 um pore size) insert were co-cultured with different dose of SR4987 and SR4987PTX seeded on the bottom well and cultured for 5 days. The Figure shows that SR4987PTX at 1:5 ratio was able to strongly inhibit B16 proliferation as visualized by crystal violet staining of B16 in the transwell line (A) and on the membrane line (B). In line (C), photos of cultured B16 cells not stained upon co-culture or not with SR4987 and SR4987PTX (20x magnifications). Figure S5. Rosette formed by B16 + SR4987 and B16 + SR4987PTX and their ultrastructural analysis by TEM. In (A) and (B) rosette formed by SR4987GFP + B16 photographed under light and fluorescence microscopy respectively. (C-F) four different panels at TEM showing the interactions between untreated SR4987GFP and B16 cells. Note that B16 cells bound SR4987GFP appear healthy (C); the presence of area of contact between B16 and SR4987 is evident; junctional-like structure and a gap-like junction (D,E) and even nanotubule structure (F) are seen (white arrows). In (G) and (H) rosette formed by SR4987GFP-PTX + B16. Note that most of B16 cells appeared damaged and necrotic under light (G) and fluorescence (H) microscopy observation. In (I-L) four different panels at TEM showing the interaction between SR4987GFP-PTX and B16. The B16 cells bound to SR4987GFP-PTX show varying degrees of degeneration, some cells seemed even emptied of their cytoplasm (I) and, similarly to untreated SR4987, area of contact (white arrows) can be observed (J,K). In (L) a microvescicole that arise from SR4987PTX is photographed. Figure S6. Metastasis in the lung of control mice and after treatment with PTX, SR4987 and SR4987PTX. On day 21 after B16 cells injection, mice were euthanized, lungs removed and fixed in Bouin’s solution. In the figure photos of the lungs, taken under a dissecting stereomicroscope. Note the presence of several melanoma lung nodules (black arrows) in control and SR4987 treated mice. In contrast, at both dosage of SR4987PTX injected, the lung nodules are very few and are significantly less than those detected in PTX treated mice. Figure S7. Chemiotaxis of SR4987 and SR4987PTX in response to B16-CM and expression of the SDF-1 receptors CXCR4 and CXCR7. In (A) migration of SR4987PTX and SR4987 placed on the top of transwell membrane insert and stimulated by different dilution of B16-CM. No significant chemotaxis activity of both SR4987 and SR4987PTX is stimulated by B16-CM at every dilutions tested. Note that the basal migration of SR4987PTX is around three fold lower than untreated SR4987 (* p < 0.05). In (B) is showed FC analysis of SR4987 for the expression of the SDF-1 receptors CXCR4 and CXCR7, indicating the high positivity of the cells particularly for CXCR4 (>90 %), CXCR7 is less expressed (18 %). (DOC 26 kb