Purging of human breast cancer cells from stem cell products with an adenovirus containing p53.

Tumor cell contamination of stem cell products can contribute to tumor relapse following high-dose chemotherapy and stem cell rescue. Numerous techniques have been used to remove the tumor cells from stem cell products with the objective of prolonging relapse-free survival. However, to date these techniques have been relatively ineffectual and/or toxic to hematopoietic stem and progenitor cells. The differential infectivity of adenovirus (Adv) vectors for breast cancer cells, compared with hematopoietic cells, has suggested that Adv-p53 might provide an effective purging strategy. To facilitate the use of Adv-p53 as a clinical strategy, we undertook studies to determine the parameters necessary for optimal stem cell product purging. The parameters studied were the particle number to nucleated cell ratio, the duration of coincubation, the incubation volume, and the presence or absence of hematopoietic progenitor cells. We have found that these parameters are interdependent and conclude that a 4-hour coincubation with an Adv-p53 particle to nucleated cell ratio of 2000:1 with 2 x 10(8) nucleated cells/mL is optimal for tumor cell purging. Furthermore, this appeared to be a safe procedure, with total loss of clonogenic growth of breast cancer cells as well as no significant effect on progenitor cell function as determined by granulocyte-macrophage colony-forming unit assays.

Adenovirus p53 purging for human breast cancer stem cell products.

Tumor cell (TC) contamination of stem cell products can contribute to relapse after high dose chemotherapy and stem cell rescue. A new purging technology using replication-deficient recombinant adenovirus (Adv) containing the p53 tumor suppressor gene (Adv-p53) has been suggested to reduce tumor contamination of autologous stem cell product. We demonstrate herein a safe and effective Adv-p53 purging procedure using four human breast cancer TC lines. Multiple parameters need to be achieved to successfully purge stem cell products, including a high cell:virus ratio, a small incubation volume, a long incubation time and 37 degrees C rather than room temperature. These parameters are all interrelated and equally important for the inhibition of TC clonogenic growth. In our studies, we also observed that Adv could nonspecifically inhibit TC clonogenic growth, although Adv-p53 treatment led to a significantly greater inhibition of clonogenic growth by cells expressing mutated p53. The presence of peripheral stem cell (PSC) products was found to decrease the effect of Adv-p53 on TC clonogenic growth, suggesting that PSC products could compete with TC for infection by recombinant Adv. However, X-Gal staining after incubation with Adv containing-galactosidase demonstrated that PSC products were 2, 000-fold more resistant to Adv infection than TC. We conclude that a 4-hour incubation of stem cell products (2 x 10(8)/ml) with 4 x 10(11) Adv-p53 particles is sufficient to completely purge TC with no effect on hematopoietic cell function.

Hematopoietic augmentation by a beta-(1,4)-linked mannan.

CARN 750 (injectable acemannan) is a polydispersed beta-(1,4)-linked acetylated mannan isolated from the Aloe barbadensis plant. It has multiple therapeutic properties including activity in wound repair and as a biological agent for the treatment of neoplasia in animals as well as the ability to activate macrophages. We report herein that CARN 750 directly or indirectly has significant hematoaugmenting properties. We observed that the subcutaneous administration of CARN 750 significantly increases splenic and peripheral blood cellularity, as well as hematopoietic progenitors in the spleen and bone marrow as determined by the interleukin-3-responsive colony-forming unit culture assay and the high-proliferative-potential colony-forming-cell (HPP-CFC) assay (a measure of primitive hematopoietic precursors) in myelosuppressed (7 Gy) C57BL/6 mice. The greatest hematopoietic effect was observed following sublethal irradiation in mice receiving 1 mg CARN 750/ animal, with less activity observed at higher or lower doses. Further, CARN 750, following daily injection, has activity equal to or greater than the injection of an optimal dose of granulocyte-colony-stimulating factor (G-CSF) in myelosuppressed mice. In this comparison, significantly greater activity was observed in the splenic and peripheral blood cellularity, and in the frequency and absolute number of splenic HPP-CFC as compared to the mice receiving G-CSF at 3 micrograms/animal. CARN 750, when administered to myelosuppressed animals, decreased the frequency of lymphocytes with a concomitant significant increase in the frequency of polymorphonuclear leukocytes (PMN). However, owing to the increased cellularity, a significant increase in the absolute number of PMN, lymphocytes, monocytes and platelets was observed, suggesting activity on multiple cell lineages. The latter is the primary difference in activity as compared to G-CSF which has activity predominantly on PMN.

In vivo haemoprotective activity of tetrapeptide AcSDKP combined with granulocyte-colony stimulating factor following sublethal irradiation.

We report that acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), which removes progenitor cells from cell cycle, in combination with granulocyte-colony stimulating factor (G-CSF) can significantly improve myelorestoration following irradiation (7 Gy). Peripheral blood, spleen and bone marrow (BM) cell recovery and progenitor cell reconstitution [IL-3-responsive colony-forming cells (CFC) and high proliferative potential colony-forming cells (HPP-CFC)] were studied. Studies on the optimal schedule of AcSDKP administration revealed maximal effects on progenitor cells when AcSDKP was administered as a continuous infusion for 3 d starting 24 h prior to irradiation and used in combination with G-CSF. The numbers of CFC and HPP-CFC in the BM were significantly increased following irradiation in mice receiving AcSDKP and G-CSF as compared to either drug alone. The numbers of CFC in the spleen were significantly increased in mice receiving AcSDKP and G-CSF on days 10 and 14 as compared to AcSDKP alone, but not G-CSF. Similarly, CFC and HPP-CFC in the spleen were significantly increased in mice receiving AcSDKP and G-CSF on day 18 as compared to mice receiving PBS and G-CSF. These studies suggest that AcSDKP in combination with G-CSF may have potential for the protection of progenitor cells in patients undergoing intensive chemo- and/or radiotherapy.

In vivo protective effects of tetrapeptide AcSDKP, with or without granulocyte colony-stimulation factor, on murine progenitor cells after sublethal irradiation.

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) demonstrated hemato-protective activity in mice after sublethal irradiation (7 GY). Bone marrow interleukin-3 (IL-3)-responsive colony-forming cells (CFC and high proliferative potential colony-forming cells (HPP-CFC) were significantly (p < 0.05) increased by day 10 after irradiation in mice receiving a continuous infusion of 1000 ng/day of AcSDKP compared to irradiated control mice. The maximum protective effect for bone marrow progenitors was achieved when AcSDKP was administered for 3 days beginning 24 hours before irradiation. Other dosages and schedules in relationship to irradiation were less active. Further, when granulocyte colony-stimulating factor (G-CSF) was administered for 10 days beginning 24 hours before irradiation. Other dosages and schedules in relationship to irradiation were less active. Further, when granulocyte colony-stimulating factor (G-CSF) was administered for 10 days after AcSDKP infusion in irradiated mice, significantly increased numbers of IL-3 responsive CSF-only control mice. In addition, platelets were significantly (p < 0.05) increased in mice receiving AcSDKP and G-CSF on days 18 and 21 after irradiation compared with mice receiving G-CSF alone. We conclude that ACSDKP has a radioprotective effect in vivo for progenitor cells, and that time of initiation and duration of AcSDKP administration relative to irradiation are crucial for these effects. Further, AcSDKP has a significant additive protective effect not only for progenitor cells but also for platelets when given in combination with G-CSF. We suggest that these in vivo observations provide a basis on which to design optimal clinical hypothesis and protocols.

Studies on optimal dose and administration schedule of a hematopoietic stimulatory beta-(1,4)-linked mannan.

Several complex carbohydrates have been found to significantly stimulate hematopoiesis. CARN 750, a polydispersed beta-(1,4)-linked acetylated mannan isolated from the Aloe vera plant, has been shown to have activity in wound repair, to function as a antineoplastic, and to activate macrophages. We report, herein, the hematoaugmenting properties of CARN 750 and its optimal dose and timing of administration in an animal model of irradiation-induced myelosuppression. We observed that subcutaneous injections of 1 mg/animal of CARN 750 had equal or greater stimulatory activity for white blood cell (WBC) counts and spleen cellularity as well as on the absolute numbers of neutrophils, lymphocytes, monocytes and platelets than did higher or lower doses of CARN 750 or an optimal dose of granulocyte-colony stimulating factor (G-CSF). Hematopoietic progenitors, measured as interleukin-3-supported colony forming units-culture (CFU-C) and high proliferative potential colony-forming cells (HPP-CFC) assays, were similarly increased by CARN 750 in the spleen but not in the bone marrow. The frequency of splenic HPP-CFCs and absolute number of splenic HPP-CFCs and CFU-Cs were optimally increased by 1 mg/animal of CARN 750. In contrast, bone marrow cellularity, frequency and absolute number of HPP-CFCs and CFU-Cs had as a dosage optimum 2 mg/animal of CARN 750. These parameters were similarly increased by G-CSF. In studies to determine the optimal protocol for the administration of CARN 750 we found that the hematopoietic activity of CARN 750 increased with the frequency of administration. The greatest activity in myelosuppressed mice was observed for all hematopoietic parameters except the platelet number in mice receiving daily administration of 1 mg/animal of CARN 750 with activity equal to or greater than G-CSF.

Interleukin-12 enhances peripheral hematopoiesis in vivo.

Interleukin 12(IL-12) is a cytokine that supports the proliferation and activation of cytotoxic T lymphocytes and natural killer (NK) cells. Recent evidence has suggested that IL-12 also has hematopoietic activities in vitro. We report studies that show that IL-12 has significant in vivo hematopoietic stimulating activity that includes enhancement of peripheral (splenic) hematopoiesis and mobilization of hematopoietic progenitor cells to the peripheral circulation. A single injection of recombinant murine IL-12 significantly reduced the number of bone marrow (BM) colony-forming unit granulocyte-macrophage (CFU-GM) in a time-dependent manner, while concomitantly stimulating high proliferative potential. In contrast, splenic CFU-GM and HPP were increased in a time- and dose-dependent manner. Chronic administration of IL-12 resulted in significant splenic hyperplasia with increased progenitor cells, increased circulating progenitor cells, and BM hypoplasia with decreased progenitor cells. These data show that IL-12 has significant in vivo hematopoietic effects that include the ability to mobilize progenitor cells to the peripheral circulation, which may prove to be of significant benefit for peripheral blood stem cell transplantation. Thus, IL-12 has potential to be an important agent for clinical transplantation because of its hematopoietic mobilization and its previously shown immune augmenting and therapeutic activities. This combination of hematopoietic and immune functions is unique and not achievable with currently used hematopoietic growth factors.