- Top of page
- MATERIALS AND METHODS
- CONFLICT OF INTEREST
BCG immunotherapy for nonmuscle-invasive bladder cancer has been successfully established for decades and intravesical instillation of Mycobacterium bovis BCG has been used to prevent recurrence of high-risk nonmuscle-invasive bladder cancer and to treat carcinoma in situ. Although the detailed mechanism has not been fully elucidated, live BCG has been established as a tuberculosis vaccine since 1921 and recognised to stimulate both innate and specific immunity to tuberculosis in human hosts. In cases of nonmuscle-invasive bladder cancer, BCG has been thought to act directly and/or indirectly on the anticancer immune system as a potent adjuvant, and to provide long lasting immune protection that prevents recurrence of nonmuscle-invasive bladder cancer [1,2]. Although immunotherapy using live BCG has been recognised as an effective tool, fatal adverse events such as systemic dissemination of BCG cannot be precluded . To avoid such unfavourable events, it is necessary to develop a more active and less toxic immunotherapeutic agent.
Mycobacterial cell walls (CWs) consist of highly characteristic hydrophobic molecules, such as mycoloyl glycolipids, mannose containing lipoglycans and CW skeleton, most of which stimulate Th-1 type immune responses. BCG survives within the cytoplasm of dendritic cell and plays a crucial role as an immunostimulant for months to years, due to its hydrophobic and unique molecular properties that contribute to preventing BCG from being digested by phagosome–lysosome fusion in dendritic cells. As the CW components of mycobacteria have been reported to stimulate antitumour responses through production of TNF-α, interleukin 12, and interferon γ in experimental animal systems [4–6], we have examined the effect of a BCG-CW preparation on bladder tumour generation with MBT-2 cells in mice. However, despite that the immunotherapeutic potential of BCG-CW by initiating the inside-outside signalling pathway through membrane-bound CD14/Toll-like receptor (TLR) 2 has been reported [7–10], this application is hampered by the unfavourable physicochemical characteristics of BCG-CW . Both negative surface charge and highly hydrophobic properties cause poor cellular association [12,13], which obstructs the critical step for evoking immune protection.
To overcome these unfavourable physicochemical properties of the BCG-CW preparation, octaarginine-modified liposomes (R8-liposomes) were applied as a vector to transport BCG-CW into the cytoplasm effectively. R8-liposomes were developed to transfer highly negative charged DNA molecules into the cytoplasm by macropinocytosis [14–16]. R8-liposomes resemble an envelope-type virus and their surface-modification by anchored R8, a characteristic and efficient cell-penetrating peptide . The enhanced ability of cellular association of stearyl R8-liposomes incorporating BCG-CW (R8-liposome-BCG-CW) was shown using a bladder cancer cell line (MBT-2) derived from C3H/HeN mouse in vitro. We also investigated whether R8-liposome-BCG-CW induced antitumour effects and long-term immunoprotection using a s.c. bladder cancer model that we have previously used to show the adjuvant effects of BCG [18,19].
MATERIALS AND METHODS
- Top of page
- MATERIALS AND METHODS
- CONFLICT OF INTEREST
For the in vitro study, BCG-CW was prepared. The CW fraction was prepared from the heat-killed cells of Mycobacterium bovis BCG Tokyo 172. Briefly, bacteria were cultivated on the surface of Sauton medium for 9 days at 37 °C and autoclaved at 121 °C for 15 min and centrifuged. The cells were re-suspended in deionized water and passed three times through a French Pressure Cells (5501-MF, Ohtake Works, Tokyo, Japan) at a pressure of 180 MPa to break the cells into small fragments. The unbroken cells were removed by centrifugation twice at 6760g for 20 min at 25 °C. In addition, the CW fraction was separated from the supernatant by ultra-centrifugation at 18 000g for 1 h at 25 °C, and then lyophilized for storage. The CW fraction thus obtained contained mainly the CW skeleton, arabinogalactan peptidoglycan mycolate complex, cord factor (trahalose 6,6′-dimycolate), lipomannan, phosphatidyl inositol di- and hexamannosides, and CW-bound proteins. The crude cell wall fraction (BCG-CW) showed sizes with 500–700 nm at median diameter.
For the preparation and characterization of R8-liposomes incorporating BCG-CW, egg phosphatidylcholine and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Sulforhodamine B, fluorescein-5-thiosemicarbazide (FTSC), Lysotracker Red, and Hoechst 33342 were purchased from Molecular Probes (Eugene, OR, USA). Stearylated-octaarginine (STR-R8) was synthesized and purified as described previously . R8-liposomes incorporating varying amounts of BCG-CW were prepared of egg phosphatidylcholine/cholesterol/STR-R8 (7:3:0.08, molar ratio). The liposomes were prepared by the lipid-film hydration method described previously . Briefly, each lipid was dissolved in chloroform/methanol (2:1, v/v) in a round-bottom flask, and BCG-CW suspended in the same solvent was added. The thin lipid film containing BCG-CW was then obtained by evaporating the solvent with a rotary evaporator under reduced pressure. The lipid film was hydrated by adding 1 mL of 10 mm PBS and shaking the flask for 20 min at 65 °C. The liposomes prepared were extruded through a polycarbonate membrane filter with a pore size of 0.4 µm using a hand extruder (Avanti Polar Lipids, Alabaster, AL, USA). Unincorporated components, which were dissociated from BCG-CW, were removed from the liposomes by ultracentrifugation at 91 000g for 30 min at 4 °C (Himac CS150 GX, Hitachi, Japan). Finally, the liposomes were resuspended in PBS and kept at 4 °C until use.
For the uptake and intracellular localization studies, fluorescent-labelled liposomes were prepared. The terminal oxidizable carbohydrate moieties exposed on the surface of BCG-CW were labelled with FTSC after periodate oxidation as described previously . Liposome formulations were prepared as described above using FTSC-labelled BCG-CW. The aqueous phase of liposomes was also labelled with sulforhodamine B. The diameter, polydispersity index, and ζ-potential of the prepared liposomes were measured by dynamic light scattering at 25 °C with a Malvern Nano Zetasizer (Malvern, Worcester, UK).
Bladder tumour cells (MBT-2) derived from C3H/HeN mice were obtained from RIKEN, Tsukuba, Japan, and maintained at 37 °C in air with 5% CO2 in RPMI 1640 medium supplemented with 10% fetal calf serum.
To investigate the cellular association of R8-liposomes containing BCG-CW, MBT-2 cells were incubated in serum-free RPMI 1640 medium containing either unmodified liposomes or R8-liposomes (both liposomes incorporating BCG-CW) at a final concentration of 0.1 mm lipids for 1 h at 37 °C, followed by three washes with ice-cold PBS. Cells were trypsinized, washed, and then suspended in 0.5 mL of FACS buffer. After being passed through a nylon mesh, the cells were analysed by flow cytometry.
To investigate the cellular interaction and uptake of R8-liposome-BCG-CW, the MBT-2 cells were treated with double-labelled R8-liposome-BCG-CW (FTSC-labelled glycolipid moiety and rhodamine-labelled aqueous phase of R8-liposome, with a final concentration of 0.1 mm lipid) in serum-free RPMI 1640 medium at 37 °C for 1 h. The cells were then washed three times with ice-cold PBS and analysed by confocal laser scanning microscopy (LSM510 Meta, Carl Zeiss). To investigate the intracellular fate of the BCG-CW incorporated into the R8-liposomes, the MBT-2 cells were treated with R8-liposome containing FTSC-labelled BCG-CW at 37 °C for 1 h. At 30 min before observation, the endosome/lysosome compartments were stained with 75 nm Lysotracker Red. Nuclei were stained with Hoechst 33342 in the last 10 min of incubation. After incubation, the cells were washed three times with ice-cold PBS and were directly analysed.
For the in vivo study, female C3H/HeN mice (7-week-old) were used. The mice were housed in plastic cages and maintained under standard conditions of temperature, humidity, and a 12:12-h light-dark cycle daily. Mice had free access to a standard diet and water. The Guide for the Care and Use of Laboratory Animals of Tsukuba University was followed at all times.
MBT-2 cells were trypsinized and washed twice with PBS. MBT-2 cells (7 × 105) were inoculated into the right side back of each mouse with 100 µL of PBS alone (group A, six mice), PBS containing 1 mg BCG (group B, six mice), 1 mg BCG-CW (group C, 18 mice), 0.1 mg BCG-CW (group D, six mice), 1 mg R8-liposome-BCG-CW (group E, 18 mice), 0.1 mg R8-liposome-BCG-CW (group F, six mice), or R8-liposomes vehicle alone (group G, six mice). The six mice in group H were not treated (Table 1). The size of the growing tumour was recorded weekly. After 4 weeks, mice bearing tumours were anaesthetized, and the tumours were removed surgically.
Table 1. The study design, mice were inoculated at 0 week, evaluated at 4 weeks, re-inoculated at 6 weeks and then evaluated again at 10
|Group||N||Inoculation at 0 week||Re-inoculation at 6 weeks after initial inoculation|
|A||6||MBT-2||MBT-2 pretreated with BCG|
|B||6||MBT-2 + 1 mg BCG||MBT-2 pretreated with BCG|
|C||6||MBT-2 + 1 mg BCG-CW||MBT-2|
|C||6||MBT-2 + 1 mg BCG-CW||MBT-2 pretreated with BCG|
|C||6||MBT-2 + 1 mg BCG-CW||MBT-2 pretreated with R8-liposome-BCG-CW|
|D||6||MBT-2 + 0.1 mg BCG-CW||MBT-2 pretreated with BCG|
|E||6||MBT-2 + 1 mg R8-liposome-BCG-CW||MBT-2|
|E||6||MBT-2 + 1 mg R8-liposome-BCG-CW||MBT-2 pretreated with BCG|
|E||6||MBT-2 + 1 mg R8-liposome-BCG-CW||MBT-2 pretreated with R8-liposome-BCG-CW|
|F||6||MBT-2 + 0.1 mg R8-liposome-BCG-CW||MBT-2 pretreated with BCG|
|G||6||MBT-2 + R8-liposomes||MBT-2 pretreated with BCG|
|H||6||none||MBT-2 pretreated with BCG|
For pretreatment of MBT-2 cells for re-inoculation, MBT-2 cells growing on culture dishes were washed twice with PBS, and then cultured for 24 h before re-inoculation in serum-free RPMI 1640 medium alone, containing BCG or R8-liposome-BCG-CW (0.1 mg/mL). Just before use for re-inoculation, pretreated MBT-2 cells were trypsinized and washed with PBS three times.
At 6 weeks after the primary inoculation, all mice except those in groups C and E were re-inoculated with 7 × 105 of BCG-pretreated MBT-2 cells in 100 µL of PBS to the left side back of the mice (Table 1). Mice that had been vaccinated with MBT-2 cells with 1 mg BCG-CW (group C) or 1 mg R8-liposome-BCG-CW (group E) were divided into three subgroups. Mice in these subgroups were s.c. re-inoculated with 7 × 105 of BCG or R8-liposome-BCG-CW pretreated or untreated MBT-2 cells in 100 µL of PBS to left side of the back. The size of the growing tumour was recorded at 4 weeks after re-inoculation.
Tumour size was estimated by calculating the area using the lengths of the two axes of the tumour with the formula S(mm2) = πab/4. The antitumour effect was assessed by comparing the size of the tumours in each group or subgroup of mice by using the Student’s t-test.
- Top of page
- MATERIALS AND METHODS
- CONFLICT OF INTEREST
The R8-liposomes incorporated with CWs derived from Mycobacterium bovis BCG Tokyo 172 (BCG-CW) used in this study were uniform of ≈230 nm in particle size with a positively charged surface (Table 2).
Table 2. The properties of the prepared R8-liposomes. The polydispersity index (P.D.) reflects the distribution of particle sizes, ranging from 0.0 for entirely monodispersed particles up to 1.0 for heterogeneous particles
|Liposome formulation||Mean (sd)particle size, nm||P.D.||Mean (sd) ζ-potential, mV|
|R8-liposome + 1 mg BCG-CW||233 (35)||0.27||26.9 (4.3)|
|R8-liposome + 0.1 mg BCG-CW||232 (43)||0.29||19.9 (9.1)|
|R8-liposome (with no BCG-CW)||270 (48)||0.31||29.7 (4.6)|
Flow cytometric analysis of MBT-2 cells incubated with either sulforhodamine-labelled liposomes containing BCG-CW (without R8) or sulforhodamine-labelled R8-liposome-BCG-CW for 1 h at 37 °C confirmed enhanced cellular interaction with the R8-liposomes (Fig. 1). There was fluorescently double-labelled R8-liposome-BCG-CW in the cellular membrane and cytoplasm after co-incubation with MBT-2 cell for 1 h at 37 °C, as the carbohydrate moieties exposed on the surface of BCG-CW were labelled with FTSC (green), while the aqueous phase of the liposomes were labelled with sulforhodamine (red), that indicated that BCG-CW was internalized into the cytoplasm of MBT-2 cell as a R8-liposome (Fig. 2a). Intracellular BCG-CW (green) was eventually co-localized with the endosome/lysosome compartment (red) (Fig. 2b).
Figure 1. Cellular association of R8-liposomes or unmodified liposomes incorporating BCG-CW with mouse bladder tumour (MBT-2) cells was analysed by flow cytometry. The MBT-2 cells were incubated with sulforhodamine-labelled liposomes containing BCG-CW (with no R8) or sulforhodamine-labelled R8-liposome-BCG-CW for 1 h at 37 °C. Flow cytometric analysis showed that more of the MBT-2 cells were associated with labelled R8-liposome-BCG-CW (red solid line) than that of MBT-2 cells associated with fluorescent-labelled unmodified liposomes containing BCG-CW (green dotted line), which overlapped the distribution of MBT-2 cells alone (autofluorescence).
Download figure to PowerPoint
Figure 2. a, Fluorescently double-labelled R8-liposome-BCG-CW was incubated with MBT-2 cell for 1 h at 37 °C. The carbohydrate moieties exposed on the surface of BCG-CW were labelled with FTSC (A), while the aqueous phase of liposomes were labelled with sulforhodamine (B). Visible light microscopic view of MBT-2 cell (C). BCG-CW and R8-liposomes were together distributed to the cellular membrane and cytoplasm of MBT-2 cell (D). b, The fate of internalized BCG-CW was tracked. The cell nuclei were stained with Hoechst 33342 (A). MBT-2 cells were incubated with R8-liposome incorporating single-labelled BCG-CW (B) at 37 °C for 1 h. Endosomes and lysosomes of MBT-2 cells were stained for 30 min before visualization with 75 nm Lysotracker Red (C). BCG-CW in the cytoplasm was co-localized with endosome/lysosome compartments (D).
Download figure to PowerPoint
The growth of tumours was initially determined at 2 weeks, but then tumours of MBT-2 cells mixed with 1 mg BCG (group B), 1 mg BCG-CW (group C) and 0.1 mg R8-liposome-BCG-CW (group D) regressed or vanished by 4 weeks. The numbers of mice bearing tumours at 4 weeks were all six in group A (PBS alone), three of six in group B (1 mg BCG), eight of 18 in group C (1 mg BCG-CW), all six in group D (0.1 mg BCG-CW), 15 of 18 in group E (1 mg R8-liposome-BCG-CW), none in group F (0.1 mg R8-liposome-BCG-CW,), and five in group G (R8-liposomes) at 4 weeks (Fig. 3a). The tumours that developed from MBT-2 cells mixed with 1 mg BCG (group B), 1 mg BCG-CW (group C), 1 mg R8-liposome-BCG-CW (group E) and 0.1 mg R8-liposome-BCG-CW (group F) were significantly smaller than those developed from MBT-2 cells alone (group A) as a control, at a mean (sd) size of 6 (7) mm2, 26 (50) mm2, 22 (21) mm2, and 0 mm2 compared with 290 (193) mm2, respectively (P < 0.001). There was no significant effect against tumour growth in the 0.1 mg BCG-CW (group D) and R8-liposomes with no BCG-CW (group G), with tumour sizes of 201 (136) mm2 and 359 (268) mm2, respectively. The 0.1 mg R8-liposome-BCG-CW (Group F) completely inhibited the growth of all tumours, while 0.1 mg BCG-CW with no R8-liposomes (Group D) did not (P = 0.002).
Figure 3. a, The growth of primary tumour was recorded weekly. The growth of MBT-2 tumours was suppressed when MBT-2 cells were inoculated with mixture of 1 mg BCG, 1 mg BCG-CW, 0.1 mg or 1 mg R8-liposome-BCG-CW compared with that of MBT-2 cells alone (*P < 0.001). The 0.1 mg R8-liposome-BCG-CW inhibited the growth of all tumours of MBT-2 cells by 4 weeks, while 0.1 mg BCG-CW with no R8-liposomes vector did not (**P = 0.002). b, The growth of re-challenged tumour was recorded weekly in mice that had been vaccinated with a mixture of MBT-2 cells and 1 mg BCG-CW. The growth of re-challenged tumours of BCG-pretreated MBT-2 cells was suppressed compared with that of MBT-2 cells with no pretreatment (**P = 0.004). c, The growth of the re-challenged tumours was recorded weekly in mice that had been vaccinated with a mixture of MBT-2 cells and 1 mg R8-liposome-BCG-CW. The growth of the re-challenged tumours of R8-liposome-BCG-CW- and BCG-pretreated MBT-2 cells was suppressed compared with that of MBT-2 cells with no pretreatment (*P = 0.003, **P < 0.001, respectively).
Download figure to PowerPoint
MBT-2 cells pretreated in vitro with BCG were re-inoculated at 6 weeks in to the mice. The number of mice bearing re-challenged tumours of BCG-pretreated MBT-2 cells was evaluated at 10 weeks (Table 3). Only one of six mice that had been vaccinated with a mixture of MBT-2 cells and either 1 mg BCG (group B) or 0.1 mg R8-liposome-BCG-CW (group F) developed a re-challenged tumour at 10 weeks, while all six mice with no vaccination (group H) developed re-challenged tumours (P = 0.002). Five of six mice that had been vaccinated with a mixture of MBT-2 cells and 1 mg R8-liposome-BCG-CW (group E) developed re-challenged tumours. Four of five mice that had been vaccinated with a mixture of MBT-2 cells and 1 mg BCG-CW developed re-challenged tumours.
Table 3. BCG-pretreated MBT-2 cells were re-inoculated in the vaccinated mice. The number of mice bearing re-challenged tumours of BCG-pretreated MBT-2 cells was recorded after 4 weeks from re-inoculation (at 10 weeks)
|Vaccination||Number of mice, n/N|
|MBT-2 + 1 mg BCG||1/5*|
|MBT-2 + 1 mg BCG-CW||4/5|
|MBT-2 + 1 mg R8-liposome-BCG-CW||5/6|
|MBT-2 + 0.1 mg R8-liposome-BCG-CW||1/6*|
|With no vaccination||6/6|
The numbers of mice bearing re-challenged tumours of BCG-, R8-liposome-BCG-CW-pretreated MBT-2 cells or MBT-2 cells with no pretreatment were four of five, three of three or five of five after 4 weeks from the re-inoculation, respectively, in mice that had been vaccinated with a mixture of MBT-2 cells and 1 mg BCG-CW. The growth of the re-challenged tumours of BCG-pretreated MBT-2 cells was suppressed, while that of MBT-2 cells with no pretreatment was not, with a mean (sd) size of 96 (156) mm2 compared with 396 (98) mm2, respectively (P = 0.004, Fig. 3b). The growth of the re-challenged tumours of R8-liposome-BCG-CW-pretreated MBT-2 cells was moderately suppressed, with a mean size of 207 (108) mm2.
The numbers of mice bearing re-challenged tumours of BCG-, R8-liposome-BCG-CW-pretreated MBT-2 cells or MBT-2 cells with no pretreatment after 4 weeks from the re-inoculation were five of six, three of three or six of six, respectively, in mice that had been vaccinated with a mixture of MBT-2 cells and 1 mg R8-liposome-BCG-CW. The growth of the re-challenged tumours of MBT-2 cells pretreated with 1 mg or 0.1 mg R8-liposome-BCG-CW was suppressed while that of MBT-2 cells with no pretreatment was not (Fig. 3C). The mean (sd) sizes of the tumours in each group were 54 (60) mm2 (P < 0.001), and 69 (44) mm2 (P = 0.003), respectively, compared with 309 (125) mm2.
- Top of page
- MATERIALS AND METHODS
- CONFLICT OF INTEREST
The antitumour mechanism of BCG and the role of its CW components have been gradually unveiled recently. Most of immunopotentiative activities are associated with the CW lipids of BCG , and which involve inflammation, innate and acquired immune responses. Intravesical instillation of BCG induces durable remission of nonmuscle-invasive bladder cancer and prevents recurrence.
Our previous study showed that mice that had been vaccinated with a mixture of MBT-2 cells and BCG, rejected re-challenged tumour of BCG-pretreated MBT-2 cells, but did not reject that of MBT-2 cells with no pretreatment. This suggests that the bladder cancer cell, which is usually under the condition of immune tolerance from the host, can be recognised through the presence of BCG-related molecules associated with the cancer cell by the immune system that was activated by the previous BCG therapy.
The aim of the present study was to develop a non-live bacterial agent using a CW extract of BCG (BCG-CW), which consisted mainly of essential molecules to induce immune responses, such as mycoloyl arabinogalactan peptideglycan complex (CWS), mycoloyl glycolipids and lipomannan [24–26]. However, the BCG-CW preparation itself has difficulty associating with the bladder cancer cells due to its highly hydrophobic properties, the crude CW fraction contains more hydrophobic molecules such as lipoglycan and glycophospholipids . Furthermore, because usually urothelial (cancer) cells are not phagocytic, transportation of a non-infectious molecule such as BCG-CW across the cell membrane is passively performed by endocytosis. Due to its hydrophobic properties, BCG-CW tends to form relatively large agglutinated clumps in an aqueous environment, which also obstructs its transportation by endocytosis. BCG-CW and cell membrane also electronically repel each other because both of their surfaces are negatively charged. Therefore, we hypothesized that the immune response would be evoked if we could successfully deliver BCG-CW into the cytoplasm of murine bladder cancer cells by using an appropriate vector.
R8-liposomes vector was originally developed as an efficient non-viral vehicle to transfer gene plasmids into cells by macropinocytosis [15,16]. Experiments using this delivery system in vivo confirmed that they can reach various organs  and cancer tissues . The incorporation of BCG-CW into R8-liposomes circumvented the unfavourable physicochemical properties of BCG-CW; the positively charged surface of the R8-liposome-BCG-CW allowing better contact to the negatively charged cell surface. It is as small as 230 nm in diameter, therefore of a molecular size that can be transported across the cellular membrane by macropinocytosis. The present in vitro study showed that R8-liposome-BCG-CW successfully attached to the surface of MBT-2 cell and was efficiently internalized into the cytoplasm within an hour of co-incubation. Internalized BCG-CW was then distributed to the lysosome of the MBT-2 cell. Although urothelial cells do not have full antigen stimulating features because they lack the second co-stimulatory signal, antigen exposure may be feasible using gradually digested external molecules with the lysosomal enzymes, as MHC class I molecule is expressed on the surface of MBT-2 cells (data is not shown).
Thus, the R8-liposome encapsulated BCG-CW had enhanced cellular association and internalization of BCG-CW into bladder cancer cells and therefore was able to induce an antitumour immune response in the host mouse. The present experiment showed that 0.1 mg BCG-CW incorporated into R8-liposomes completely inhibited the growth of MBT-2 bladder tumour at 4 weeks, while 0.1 mg BCG-CW with no R8-liposomes did not evoke highly effective antitumour activity. The BCG-induced antitumour effect involves both innate and acquired immune responses, and therefore we expected both immunostimulatory activities. As we have previously shown that the ‘normal’ urothelial cell, which is not a professional immune cell, expressed TLR-2, -3, -4 and -9 and was able to respond directly to BCG , BCG-CW incorporated with R8-liposomes contacted the cellular surface and might induced innate immune responses through the stimulation of TLR on the bladder cancer cell. During the 4 weeks of the primary inoculation experiment, the tumours regressed or vanished in the mice that were inoculated with a mixture of MBT-2 cells and 1 mg BCG (group B), 1 mg BCG-CW (group C), or 0.1 mg R8-liposome-BCG-CW (group F). As it takes longer for immunostimulation to establish acquired immune protection such as cell-mediated immunity in the host, this phenomenon might suggest that acquired-specific immune protection plays an important role in BCG immunotherapy in later stages, which is similar to the immune response against infection by the host.
As it is known that BCG immunotherapy provides long lasting immune protection against recurrence of nonmuscle-invasive bladder cancer, the results of the re-challenge experiment produced important findings related to immune memory induced by BCG (Fig. 3b,c). The suppressive growth of re-challenged tumour was seen only if BCG- or R8-liposome-BCG-CW-pretreated MBT-2 cells were re-inoculated in to the vaccinated mice. As CD4+ T cell lines from BCG-treated mice showed a specific immune response against bladder cancer cells that were co-cultured with BCG , immune memory induced in vaccinated mice must recognise the MBT-2 cancer cell through the BCG-CW antigen exposure on it. Thus, immunization using BCG-CW was effective due to the modification with R8-liposomes vector, which overcomes the unfavourable physicochemical properties of BCG-CW and improves cellular association.
In the present study, antitumour activity was induced better by 0.1 mg R8-liposome-BCG-CW than by 1 mg R8-liposome-BCG-CW. In the primary challenge experiments, although both 0.1 mg and 1 mg R8-liposome-BCG-CW clearly suppressed tumour growth, 0.1 mg R8-lipsome-BCG-CW inhibited tumour growth completely by 4 weeks (Fig. 3a). In the re-challenge experiments showing the impact of vaccination, mice vaccinated with 0.1 mg R8-liposome-BCG-CW had a better effect on tumour growth than those vaccinated with 1 mg R8-liposome-BCG-CW. The phenomenon of nonlinear dose–response is known in immune responses. One of the reasons that might explain this in the present experiments may be the incidence of oversaturated BCG-CW in the cytoplasm. Because transportation of BCG-CW into the cytoplasm is performed efficiently by R8-liposomes vector as shown in vitro, the amount of BCG-CW in the cytoplasm may easily exceed the appropriate concentration to activate the immunological responses of the cell. A dependency study of R8-liposome-BCG-CW is planned to elucidate the optimum concentration for BCG-CW incorporation.
In conclusion, the immunotherapeutic potential of BCG-CW was enhanced by improving cellular association using an R8-liposomes delivery system. Development of this non-live bacterial agent may contribute to providing a more active and less toxic tool as a substitute for live BCG in immunotherapy against nonmuscle-invasive bladder cancer in the future.